Libraries of genetic packages comprising novel HC CDR1, CDR2, and CDR3 and novel LC CDR1, CDR2, and CDR3 designs

ABSTRACT

Provided are compositions and methods for preparing and identifying antibodies having CDR3s that vary in sequence and in length from very short to very long which in certain embodiments may bind to a carbohydrate moiety or the active site of an enzyme. Libraries coding for antibodies with the CDR3s are also provided. The libraries can be provided by modifying a pre-existing nucleic acid library.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/177,199, filed Jun. 8, 2016, which is a continuation application of U.S. patent application Ser. No. 12/989,307, filed Mar. 10, 2011, which is a national phase application under 35 U.S.C. § 371 of International Application No. PCT/US2009/041688 filed Apr. 24, 2009, which claims priority to U.S. Application Ser. No. 61/047,529, filed on Apr. 24, 2008. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

BACKGROUND

It is now common practice in the art to prepare libraries of genetic packages that individually display, display and express, or comprise a member of a diverse family of peptides, polypeptides or proteins and collectively display, display and express, or comprise at least a portion of the amino acid diversity of the family. In many common libraries, the peptides, polypeptides or proteins are related to antibodies (e.g., single chain Fv (scFv), Fv, Fab, whole antibodies or minibodies (i.e., dimers that consist of V_(H) linked to V_(L))). Often, they comprise one or more of the CDRs and framework regions of the heavy and light chains of human antibodies.

Peptide, polypeptide or protein libraries have been produced in several ways. See, e.g., Knappik et al., J. Mol. Biol., 296, pp. 57-86 (2000), which is incorporated herein by reference. One method is to capture the diversity of native donors, either naive or immunized. Another way is to generate libraries having synthetic diversity. A third method is a combination of the first two. Typically, the diversity produced by these methods is limited to sequence diversity, i.e., each member of the library has the same length but differs from the other members of the family by having different amino acids or variegation at a given position in the peptide, polypeptide or protein chain. Naturally diverse peptides, polypeptides or proteins, however, are not limited to diversity only in their amino acid sequences. For example, human antibodies are not limited to sequence diversity in their amino acids, they are also diverse in the lengths of their amino acid chains.

SUMMARY

For antibodies, heavy chain diversity in length occurs, for example, during variable region rearrangements. See e.g., Corbett et al., J. Mol. Biol., 270, pp. 587-97 (1997). The joining of V genes to J genes, for example, results in the inclusion of a recognizable D segment in CDR3 in about half of the heavy chain antibody sequences, thus creating regions encoding varying lengths of amino acids. D segments are more common in antibodies having long HC CDR3s. The following also may occur during joining of antibody gene segments: (i) the end of the V gene may have zero to several bases deleted or changed; (ii) the end of the D segment may have zero to many bases removed or changed; (iii) a number of approximately random bases may be inserted between V and D or between D and J; and (iv) the 5′ end of J may be edited to remove or to change several bases. These rearrangements result in antibodies that are diverse both in amino acid sequence and in length. HC CDR3s of different lengths may fold into different shapes, giving the antibodies novel shapes with which to bind antigens. The conformations depend on both the length and the sequence of the CDR3. It should be remembered that a HC CDR3 of length 8, for example, and of any sequence cannot adequately mimic the behavior of a CDR3 of length 22, for example.

Libraries that contain only amino acid sequence diversity are, thus, disadvantaged in that they do not reflect the natural diversity of the peptide, polypeptide or protein that the library is intended to mimic. Further, diversity in length may be important to the ultimate functioning of the protein, peptide or polypeptide. For example, with regard to a library comprising antibody regions, many of the peptides, polypeptides, proteins displayed, displayed and expressed, or comprised by the genetic packages of the library may not fold properly or their binding to an antigen may be disadvantaged, if diversity both in sequence and length are not represented in the library.

An additional disadvantage of such libraries of genetic packages that display, display and express, or comprise peptides, polypeptides and proteins is that they are not focused on those members that are based on natural occurring diversity and thus on members that are most likely to be functional and least likely to be immunogenic. Rather, the libraries, typically, attempt to include as much diversity or variegation as possible at every CDR position. This makes library construction time-consuming and less efficient than necessary. The large number of members that are produced by trying to capture complete diversity also makes screening more cumbersome than it needs to be. This is particularly true given that many members of the library will not be functional or will be non-specifically sticky.

In addition to the labor of constructing synthetic libraries is the question of immunogenicity. For example, there are libraries in which all CDR residues are either Tyr (Y) or Ser (S). Although antibodies (Abs) selected from these libraries show high affinity and specificity, their very unusual composition may make them immunogenic. The present invention is directed toward making Abs that could well have come from the human immune system and so are less likely to be immunogenic. The libraries of the present invention retain as many residues from V-D-J or V-J fusions as possible. To reduce the risk of immunogenicity, it may be prudent to change each non-germline amino acid in both framework and CDRs back to germline to determine whether the change from germline is needed to retain binding affinity. Thus, a library that is biased at each varied position toward germline will reduce the likelihood of isolating Abs that have unneeded non-germline amino acids.

Abs are large proteins and are subject to various forms of degradation. One form of degradation is the deamidation of Asn and Gln residues (especially in Asn-Gly or Gln-Gly) and the isomerization of Asp residues. Another form of degration is the oxidation of methionines, tryptophan, and cysteine. Another form of degradation is the cleavage of Asp-Pro dipeptides. Another form of degradation is the formation of pyroglutamate from N-terminal Glu or Gln. It is advantageous to provide a library in which the occurance of problematic sequences is minimized.

Provided are libraries of vectors or packages that encode members of a diverse family of human antibodies comprising heavy chain (HC) CDR3s that are between about 3 amino acids in length to about 35 amino acids in length. The HC CDR3s may also, in certain embodiments, may be rich in Tyr (Y) and Ser (S) and/or comprise diversified D regions and/or comprise extended JH regions. For example, the HC CDR3s may contain greater than about 40% (e.g., between about 43% and about 80%; e.g., greater than about 40% but less than about 100%) Y and/or S residues, e.g., as provided in the examples herein. Also provided are focused libraries comprising such HC CDR3s. Also provided are designs for HC CDR1, HC CDR2, and a library of VKIII A27 with diversity in the CDRs. A library of vectors or packages that encode members of a diverse family of human antibodies comprising HC CDR3s described herein can further have diversity at one or more (e.g., at one, two or three) of HC CDR1, HC CDR2, LC CDR1, LC CDR2, and LC CDR3. For example, the library can have diversity at one or more (e.g., at one, two or three) of HC CDR1, HC CDR2, LC CDR1, LC CDR2, and LC CDR3 as described herein.

A diversified D region is a D region into which one or more amino acid changes have been introduced (e.g., as compared to the sequence of a nautrally occurring D region; for example, a stop codon can be changed to a Tyr residue).

An extended JH region is a JH region that has one or more amino acid residues present at the amino terminus of the framework sequence of the JH region (e.g., amino terminal to FR4 sequences, e.g., which commence with WGQ . . . ). For example, JH1 is an extended JH region. As other examples, JH2, JH3, JH4, JH5, and JH6 are extended JH regions.

Provided also are methods of making and screening the above libraries and the HC CDR3s and antibodies obtained in such screening. Compositions and kits for the practice of these methods are also described herein.

In some aspects, the disclosure features a focused library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins (e.g., a diverse family of antibodies) and collectively display, display and express, or comprise at least a portion of the diversity of the family, wherein the vectors or genetic packages comprise variegated DNA sequences that encode a heavy chain (HC) CDR3 selected from the group consisting of:

-   -   (a) a HC CDR3 that is about 3 or about 4 or about 5 amino acids         in length;     -   (b) a HC CDR3 that is about 23, about 24, about 25, about 26,         about 27, about 28, about 29, about 30, about 31, about 32,         about 33, about 34 or about 35 amino acids in length (e.g.,         about 23 to about 35 amino acids in length); and     -   c) a HC CDR3 that is from about 6 to about 20 amino acids in         length (e.g., about 6, about 7, about 8, about 9, about 10,         about 11, about 12, about 13, about 14, about 15, about 16,         about 17, about 18, about 19, or about 20 amino acids in         length);

wherein the HC CDR3 comprises amino acids from a D region (e.g., a diversified D region) (or fragment thereof (e.g., 3 or more amino acids of the D region, e.g., diversified D region)) or a JH region (e.g., an extended JH region).

In some embodiments, the HC CDR3 is enriched in Tyr (Y) and Ser (S) (e.g., greater than 40% of the residues of the HC CDR3 are Y and/or S).

In some embodiments, the library (e.g., the vectors or genetic packages thereof) comprises a D region or a fragment of a D region (e.g., wherein the D region is adjacent to a JH region).

In some embodiments, the library comprises a JH region, e.g., an extended JH region.

In some embodiments, the HC CDR3 comprises amino acids from a D region or a fragment of a D region (e.g., wherein the D region is adjacent to a JH region).

In some embodiments, the D region is selected from the group consisting of D2-2 (RF 2), D2-8(RF 2), D2-15(RF 2), D2-21(RF 2), D3-16(RF 2), D3-22 (RF 2), D3-3 (RF-2), D3-9 (RF 2), D3-10 (RF 2), D1-26 (RF 3), D4-11 (RF 2), D4-4 (RF 2), D5-5 (RF 3), D5-12 (RF 3), D5-18 (RF 3), D6-6 (RF1), D6-13 (RF 1), and D6-19 (RF 1).

In some embodiments, the HC CDR3 comprises amino acids from a JH region. The JH region may be an extended JH region. In some embodiments, the extended JH region is selected from the group consisting of JH1, JH2, JH3, JH4, JH5, and JH6. In some embodiments, the JH region may be enriched in Y and/or S residues, for example, it may contain greater than about 40% (e.g., between about 43% and about 80%; e.g., greater than about 40% but less than about 100%) Y and/or S residues.

In some embodiments, the D region comprises one or more cysteine (Cys) residues and in some embodiments, the one or more Cys residues are held constant (e.g., are not varied).

In some embodiments, the HC CDR3 (e.g., the DNA encoding the HC CDR3) comprises one or more filling codons between FR3 and the D region and each filling codon is individually NNK, TMY, TMT, or TMC (TMY, TMT, or TMC encode S or Y).

In some embodiments, the HC CDR3 (e.g., the DNA encoding the HC CDR3) comprises one or more filling codons between the D region and JH and each filling codon is individually NNK, TMY, TMT, or TMC.

In some embodiments, the library (e.g., the vectors or genetic packages of the library) further comprises a HC CDR1, HC CDR2, and/or a light chain and also comprises diversity in the HC CDR1, HC CDR2, or light chain comprises diversity in HC CDR1 and/or HC CDR2, and/or a light chain (e.g., kappa or lambda light chain) (respectively). For example, HC CDR3 diversity can be constructed in the background of diversity in HC CDR1, HC CDR2, and/or light chains. For example, the light-chain diversity may be encoded in the same DNA molecule as the HC diversity or the LC and HC diversities may be encoded in separate DNA molecules.

In some aspects, the disclosure features a library comprising a HC CDR3 that is 3, 4, or 5 amino acids in length, wherein the CDR3 comprises amino acids from a JH region (e.g., extended JH region) or from a D region (e.g., a diversified D region) (or fragment thereof (e.g., 3 or more amino acids of the D region, e.g., diversified D region)) joined to the FR4 portion of a JH region.

In some embodiments, the HC CDR3 is from a D region joined to the FR4 portion of a JH region and comprises a trimer, a tetramer, or a pentamer, wherein the trimer, tetramer, or pentamer does not comprise a cysteine residue.

In some embodiments, the HC CDR3 is from a D region joined to the FR4 portion of a JH region and comprises a trimer, a tetramer, or a pentamer, wherein the trimer, tetramer, or pentamer does not comprise a stop codon.

In some embodiments, the D region (e.g., the DNA encoding the D region) comprises a TAG codon and the TAG codon is replaced by a codon selected from the group consisting of TCG, TTG, TGG, CAG, AAG, TAT, and GAG.

In some embodiments, the D region (e.g., the DNA encoding the D region) comprises a TAA codon and the TAA codon is replaced by a codon selected from the group consisting of TCA, TTA, CAA, AAA, TAT, and GAA.

In some embodiments, the D region (e.g., the DNA encoding the D region) comprises a TGA codon and the TGA codon is replaced by a codon selected from the group consisting of TGG, TCA, TTA, AGA, and GGA.

In some embodiments, the library further comprises diversity in HC CDR1 and/or HC CDR2, and/or a light chain (e.g., kappa or lambda light chain). For example, HC CDR3 diversity can be constructed in the background of diversity in HC CDR1, HC CDR2, and/or light chains. For example, the light-chain diversity may be encoded in the same DNA molecule as the HC diversity or the LC and HC diversities may be encoded in separate DNA molecules.

In some aspects, the disclosure provides a method of diversifying a library, the method comprising mutagenizing a library described herein.

In some embodiments, the mutagenizing comprises error-prone PCR.

In some embodiments, the mutagenizing comprises wobbling.

In some embodiments, the mutagenizing comprises dobbling.

In some embodiments, the mutagenizing introduces on average about 1 to about 10 mutations (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 mutations; e.g., base changes) per HC CDR3.

“Wobbling” is a method of making variegated DNA so that an original sequence is favored. If the original sequence had, for example, an Ala that could be encoded with GCT the mixture (0.7 G, 0.1 A, 0.1 T, 0.1 C) can be used for the first position, (0.7 C, 0.1 A, 0.1 T, 0.1 G) at the second position, and (0.7 T, 0.1 A, 0.1 G, 0.1 C) at the third. Other ratios of “doping” can be used. This allows Ala to appear about 50% of the time while V, D, G, T, P, and S occur about 7% of the time. Other AA types occur at lower frequency.

In some aspects, the present disclosure is drawn, e.g., to keeping a (purified) HC CDR1-2 repertoire, and building synthetic HC CDR3 and LC diversity.

In some embodiments, the disclosure provides a cassette for displaying a wobbled heavy chain (HC) CDR3, for example, wherein the cassette comprises the cassette shown in Table 400.

In some aspects, the present disclosure features a library in which Tyr levels are controlled in the HC CDR3. In some embodiments, the HC CDR3 regions contain about 15% or greater (e.g., about 16%, about 18%, about 20%, or greater) Tyr residues. In some embodiments, high levels (e.g., more than about 20%) of Tyr are inserted into the HC CDR3 of library members, e.g., at D regions and J stumps (or synthetic sequences corresponding thereto) that contain Tyr. In some embodiments, at leadin or DJ filler positions (or synthetic sequences corresponding thereto), Tyr is allowed, but at no more than 20%. In some embodiments, the HC CDR3 regions contain less than about 15% (e.g., about 14%, about 12%, about 10%, about 8%, about 6% or less)Tyr residues. In some embodiments, the HC leadin or DJ filler positions (or synthetic sequences corresponding thereto) contain less than about 15% (e.g., about 14%, about 12%, about 10%, about 8%, about 6% or less) Tyr residues.

In some aspects, the disclosure features a library of genetic packages that encode a human antibody heavy chain in which a parent amino-acid sequence comprises a VH sequence followed by zero to ten amino acids selected from the group consisting of (Y, S, D, L, R), followed by a human D-region or fragment of a D-region, followed by zero to ten amino acids selected from the group consisting of (Y,S,R,D,L), followed by a JH segment that comprises at least W103 onward wherein the variable DNA encoding this sequence is synthesized in a way that the parental amino-acid sequence is the most likely one (e.g., by wobbling).

In some aspects, the disclosure features a library of light chains having germline framework regions and wherein the CDRs are varied such that residues remote from the combining site or having buried side groups are held constant. In some embodiments, a method of variable DNA synthesis is used so that germline sequence is the most likely one (e.g., by wobbling).

In some aspects, the disclosure features a library of diverse members encoding antigen binding variable regions as disclosed herein.

In some aspects, the disclosure features a library of diverse members encoding HC CDR3 regions as disclosed herein. In some embodiments, the library is a library of Table 1097.

In some aspects, the disclosure features a library of diverse members, each member encoding comprising a HC CDR 3, wherein at least 1, 2, 3, 4, 5, 6, 7, or 8 positions in the HDCR3, respectively, is occupied by G, S, R, D, L, and Y in the library in the following proportions [1.0G, 0.57S, 0.46R, 0.42D, 0.36L, 0.35Y] and optionally,

the last 4 positions of HC CDR3 are represented as follows:

the parental amino acid is present at 3, 4, 5, 6, 7, 8, 10 times as likely as other amino-acid types, wherein the other amino-acid types comprise Y, S, D, R, G.

In some aspects, the disclosure features a library of diverse members, each member encoding comprising a HC CDR 3, wherein

at least one and preferably all of the first 1, 2, 3, 4, 5, 6, 7, or 8 positions in the HC CDR3, is occupied by G, S, R, D, L, and Y, in the library in the following proportions [1.0G, 0.57S, 0.46R, 0.42D, 0.36L, 0.35Y] and optionally

the last 4 positions of HCDR3 are represented as follows:

the parental amino acid is present at 3, 4, 5, 6, 7, 8, 10 times as likely as other amino-acid types, wherein the other amino-acid types comprise Y, S, D, R, G.

In some aspects, the disclosure features a library of diverse members, each member encoding a HC CDR 3, wherein the length of HC CDR3 is 10, 11, or 12 positions;

each of the first 6, 7, or 8 positions in the HC CDR3, respectively, is occupied by G, S, R, D, L, and Y in the library in the following proportions [1.0G, 0.57S, 0.46R, 0.42D, 0.36L, 0.35Y];

the last 4 positions of HCDR3 are represented as follows:

the parental amino acid is present at 3, 4, 5, 6, 7, 8, 10 times as likely as other amino-acid types, wherein the other amino-acid types comprise Y, S, D, R, G.

In some embodiments, each of the last 4 HC CDR3 positions is represented in the library as 7/12 parental, plus 1/12 each of Y, S, D, R, and G.

In some embodiments, each of the last 4 HC CDR3 positions is represented in the library as A6=7/12 A, plus 1/12 each of Y, S, D, R, and G; F7=7/12 F plus 1/12 each of Y, S, D, R, and G; D8=7/11 D plus 1/11 of Y, S, R, and G; 19=7/12 I plus 1/12 Y, S, R, D, G.

In some embodiments, the members further encode HC CDR1, HC CDR2.

In some embodiments, the members further encode Fframwork (FR) regions 1-4.

In some embodiments, the members encode HC CDR1, HC CDR2 and FR regions 1-4.

In some embodiments, the members comprise a 3-23 HC framework.

In some embodiments, the library further comprises a LC variable region.

In some embodiments, the library comprises members encoding diverse LC variable regions.

In some embodiments, the members comprising a LC variable region comprise an A27 LC framework.

In some embodiments, the library is a display library, e.g., a phage display library.

In some embodiments, the library has at least 10⁴, 10⁵ 10⁶, 10⁷, 10⁸, 10⁹ 10¹⁰, 10¹¹ diverse members.

In some aspects, the disclosure features a method of selecting a library member, comprising, contacting a library described herein with a target, allowing a member to bind to said target, and recovering the member which binds the target.

These embodiments of the present invention, other embodiments, and their features and characteristics will be apparent from the description, drawings, and claims that follow.

DETAILED DESCRIPTION

Antibodies (“Ab”) concentrate their diversity into those regions that are involved in determining affinity and specificity of the Ab for particular targets. These regions may be diverse in sequence or in length. Generally, they are diverse in both ways. However, within families of human antibodies the diversities, both in sequence and in length, are not truly random. Rather, some amino acid residues are preferred at certain positions of the CDRs and some CDR lengths are preferred. These preferred diversities account for the natural diversity of the antibody family.

According to this invention, and as more fully described below, libraries of vectors and genetic packages that encode members of a diverse family of human antibodies comprising heavy chain (HC) CDR3s that are between about 3 to about 35 amino acids in length may be prepared and used. The HC CDR3s may also, in certain embodiments, may be rich in Y and S and/or comprise diversified D regions. Also provided are focused libraries comprising such HC CDR3s.

When an immune cell constructs an antibody heavy chain, it connects a V segment to a D segment and that to a J segment. The D segment is optional and about 50% of human Abs have recognizable Ds. The cell may perform considerable editing at the junction sites (V-to-D, D-to-J, or V-to-J) both removing and adding bases, but not exactly randomly. The initially rearranged antibody is presented on the surface of the cell and if it binds an antigen (Ag), the cell is stimulated to perform somatic mutations to improve the affinity. There are hot spots encoded in the immunoglobulin germline genes so that certain places in the Ab gene are very likely to go through a particular set of mutations in search of a better binder to a persistent Ag. In nature, some of the mutations are in framework positions but most are in the complementarity determining regions (CDRs). Of particular interest is the CDR3 of the heavy chain (HC) because it shows not only a high degree of sequence diversity but also length diversity. Antibody (Ab) libraries have been built in which the CDRs are replaced with random DNA, and useful Abs have been obtained. However, some therapeutic Abs show a significant degree of antigenicity. It is possible that Abs that are closer to human germline would be less antigenic.

Definitions

For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are defined here.

The singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

The term “affinity” or “binding affinity” refers to the apparent association constant or K_(a). The K_(a) is the reciprocal of the dissociation constant (K_(d)). A binding protein may, for example, have a binding affinity of at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ and 10¹¹ M⁻¹ for a particular target molecule. Higher affinity binding of a binding protein to a first target relative to a second target can be indicated by a higher K_(A) (or a smaller numerical value K_(D)) for binding the first target than the K_(A) (or numerical value K_(D)) for binding the second target. In such cases, the binding protein has specificity for the first target (e.g., a protein in a first conformation or mimic thereof) relative to the second target (e.g., the same protein in a second conformation or mimic thereof; or a second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, or 10⁵ fold.

Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in TRIS-buffer (50 mM TRIS, 150 mM NaCl, 5 mM CaCl₂ at pH7.5). These techniques can be used to measure the concentration of bound and free binding protein as a function of binding protein (or target) concentration. The concentration of bound binding protein ([Bound]) is related to the concentration of free binding protein ([Free]) and the concentration of binding sites for the binding protein on the target where (N) is the number of binding sites per target molecule by the following equation: [Bound]=N·[Free]/((1/K _(A))+[Free]).

It is not always necessary to make an exact determination of K_(A), though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to K_(A), and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.

The term “antibody” refers to a protein that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. Heavy chain and light chain may also be abbreviated as HC and LC, respectively. The term “antibody” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab′)₂, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (de Wildt et al., Eur J Immunol. 1996; 26(3):629-39)) as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). Antibodies may be from any source, but primate (human and non-human primate) and primatized are preferred.

The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (“FR”). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, see also www.hgmp.mrc.ac.uk). Kabat definitions are used herein. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. In IgGs, the heavy chain constant region includes three immunoglobulin domains, CH1, CH2 and CH3. The light chain constant region includes a CL domain. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The light chains of the immunoglobulin may be of types, kappa or lambda. In one embodiment, the antibody is glycosylated. An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity.

One or more regions of an antibody can be human or effectively human. For example, one or more of the variable regions can be human or effectively human. For example, one or more of the CDRs can be human, e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3. Each of the light chain CDRs can be human. HC CDR3 can be human. One or more of the framework regions can be human, e.g., FR1, FR2, FR3, and FR4 of the HC or LC. For example, the Fc region can be human. In one embodiment, all the framework regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non-hematopoietic cell. In one embodiment, the human sequences are germline sequences, e.g., encoded by a germline nucleic acid. In one embodiment, the framework (FR) residues of a selected Fab can be converted to the amino-acid type of the corresponding residue in the most similar primate germline gene, especially the human germline gene. One or more of the constant regions can be human or effectively human. For example, at least 70, 75, 80, 85, 90, 92, 95, 98, or 100% of an immunoglobulin variable domain, the constant region, the constant domains (CH1, CH2, CH3, CL), or the entire antibody can be human or effectively human.

All or part of an antibody can be encoded by an immunoglobulin gene or a segment thereof. Exemplary human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the many immunoglobulin variable region genes. Full-length immunoglobulin “light chains” (about 25 KDa or about 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus. Full-length immunoglobulin “heavy chains” (about 50 KDa or about 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids). The length of human HC varies considerably because HC CDR3 varies from about 3 amino-acid residues to over 35 amino-acid residues.

Herein, the terms “D segment” and “D region” are used interchangeably and are identical. It is to be understood that these items have both DNA and amino-acid representations and that which is meant is clear from the context.

A “library” or “display library” refers to a collection of nucleotide, e.g., DNA, sequences within clones; or a genetically diverse collection of polypeptides displayed on replicable display packages capable of selection or screening to provide an individual polypeptide or a mixed population of polypeptides.

The term “package” as used herein refers to a replicable genetic display package in which the particle is displaying a polypeptide at its surface. The package may be a bacteriophage which displays an antigen binding domain at its surface. This type of package has been called a phage antibody (pAb).

A “pre-determined target” refers to a target molecule whose identity is known prior to using it in any of the disclosed methods.

The term “replicable display package” as used herein refers to a biological particle which has genetic information providing the particle with the ability to replicate. The particle can display on its surface at least part of a polypeptide. The polypeptide can be encoded by genetic information native to the particle and/or artificially placed into the particle or an ancestor of it. The displayed polypeptide may be any member of a specific binding pair e.g., heavy or light chain domains based on an immunoglobulin molecule, an enzyme or a receptor etc. The particle may be, for example, a virus e.g., a bacteriophage such as fd or M13.

The term “vector” refers to a DNA molecule, capable of replication in a host organism, into which a gene is inserted to construct a recombinant DNA molecule. A “phage vector” is a vector derived by modification of a phage genome, containing an origin of replication for a bacteriophage, but not one for a plasmid. A “phagemid vector” is a vector derived by modification of a plasmid genome, containing an origin of replication for a bacteriophage as well as the plasmid origin of replication.

In discussing oligonucleotides, the notation “[RC]” indicates that the Reverse Complement of the oligonucleotide shown is the one to be used.

Human Antibody Heavy Chain CDR3s

The heavy chain (“HC”) Germ-Line Gene (GLG) 3-23 (also known as VP-47) accounts for about 12% of all human Abs and is preferred as the framework in the preferred embodiment of the invention. It should, however, be understood that other well-known frameworks, such as 4-34, 3-30, 3-30.3 and 4-30.1, may also be used without departing from the principles of the focused diversities of this invention.

In addition, JH4 (YFDYW₁₀₃GQGTLVTVSS (SEQ ID NO:1)) occurs more often than JH3 in native antibodies. Hence, it is preferred for the focused libraries of this invention. However, JH3 (AFDIW₁₀₃GQGTMVTVSS (SEQ ID NO:2)), JH6

(SEQ ID NO: 3) (YYYYYGMDV W ₁₀₃ GQGTTVTVSS), JH1, JH2, or JH5 could be used as well. JH2 has the advantage of having RG at 105-106 instead of QG in all the other human JHs. JH3 has the disadvantage of M₁₀₈. In a collection of 1419 Abs that were ELISA positive for at least one target, we saw 17 JH1s, 31 JH2s, 452 JH3s, 636 JH4s, 32 JH5s, and 251 JH6s. If present, the double underscored portions of the JHs are considered to be part of CDR3. In Table 3, the FR4 parts of the JHs are underscored.

The frequency at which each amino-acid appeared in the HC CDR3s of these 1419 Abs was tabulated and recorded in Table 75. Note that the most common amino acid is Tyr with Gly, Asp, Ser, and Arg following in that order. Rel. Up is the relative abundance of each type compared to Cys, the least common. Rel. Down is the abundance of each type compared to Tyr, the most common. Hence the preferred amino-acid types to substitute into HC CDR3s are Y, G, D, S, and R.

Naturally, HC CDR3s vary in length. About half of human HCs consist of the components: V::nz::D::ny::JHn where V is a V gene, nz is a series of bases that are essentially random, D is a D segment, often with heavy editing at both ends, ny is a series of bases that are essentially random, and JHn is one of the six JH segments, often with heavy editing at the 5′ end. The D segments appear to provide spacer segments that allow folding of the IgG. The greatest diversity is at the junctions of V with D and of D with JH.

Corbett et al. (Corbett S J, Tomlinson I M, Sonnhammer E L, Buck D, Winter G. J Mol Biol. 1997 V270:587-97) showed that the human immune system does not insert multiple D segments and recombing D segments. Nevertheless, D segments have been selected to be good components of HC CDR3s and the present invention comprises HC CDR3 that contain more than one D segment.

Human D segments have some very strong biases. The tally of the 522 amino-acids in human D segments is Y 70 (13.4%), L 63 (12.1%), V 52 (10%), G 49 (9.4%), I 41 (7.9%), T 40 (7.7%), S 33 (6.3%), W 27 (5.2%), D 21 (4%), A 19 (3.6%), R 16 (3.1%), TAG 15 (2.9%), N 14 2.7%), Q 11 (2.1%), C 9 (1.7%), E 9 (1.7%), F 8 (1.5%), M 8 (1.5%), TGA 8 (1.5%), TAA 7 (1.3%), P 1 (0.2%), H 1 (0.2%), and K 0 (0%). There is one D (2-8 RF 1) that has an unpaired Cys but also a TGA stop codon, so it is little used. Thus, D segments are primarily hydrophobic. The frequencies of amino acids in human HC CDR3s are shown in Table 75. There are both similarities and differences in the frequencies. In HC CDR3s overall, Tyr is the most common and only Gly comes close (96% as common as Tyr). Asp (75% as common as Tyr), Ser (53% as common as Tyr). Leu, Val, and Ile are relatively common in the D segments if all the D segments are counted as equal. The immune system does not use the D segments with equal frequency. Table 77 shows the frequency of utilization of D segments. The D segments that are often used are very rich in Tyr, Gly, Ser, and Asp. Arg is not found in the most often used D segments nor is Arg encoded in any of the CDR portions of JH segments. Arg comes to prominence either by mutation of V, D, and J or in the filler regions between V and D, D and J, or V and J. In this sample, 50% of all the amino acids are Tyr, Gly, Asp, Ser, or Arg. In one embodiment of the present invention, substitutions of “parental” HC CDR3 sequences is limited to the set of amino acids consisting of Tyr, Gly, Ser, Asp, and Arg. In one embodiment of the present invention, Arg is made common in the filler regions between V and D, between D and J, or between V and J.

In the preferred libraries of this invention, both types of HC CDR3s are used. In HC CDR3s that have no identifiable D segment, the structure is V::nz::JHn (n=1,6) where JH is usually edited at the 5′ end. In HC CDR3s that have an identifiable D segment, the structure is V::nz::D::ny::JHn.

Provided herein are HC CDR3s that are between about 3 to a about 35 amino acids in length. The HC CDR3s may also, in certain embodiments, be rich in Y and S and/or comprise diversified D regions, where a D region is present. For example, the HC CDR3s may contain between about 43% and about 80% Y and/or S residues, e.g., about 43%, about 48%, about 69%, about 63%, about 71%, about 62%, about 58%, about 68%, about 80%, about 77%, or greater than about 40%, or about 40% to less than about 100%, of the residues are Y and/or S. For example, not all of the residues in the CDR3 are Y and/or S. The HC CDR3s may, in certain embodiments, comprise an extended JH region. Exemplary HC CDR3 component designs of the preferred libraries of this invention are shown and described in Examples 1, 2, and 3.

In some embodiments, diversity (e.g., in a CDR, e.g., HC CDR3, or framework region (e.g., framework region near or adjacent to a CDR, e.g., CDR3, e.g., HC CDR3) is generated to create on average about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or about 1 to about 10 mutations (e.g., base change), e.g., per CDR (e.g., HC CDR3) or framework region (e.g., framework region near or adjacent to a CDR, e.g., CDR3, e.g., HC CDR3). In some implementations, the mutagenesis is targeted to regions known or likely to be at the binding interface. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs. In the case of antibodies, mutagenesis can also be limited to one or a few of the CDRs, e.g., to make precise step-wise improvements. Likewise, if the identified ligands are enzymes, mutagenesis can provide antibodies that are able to bind to the active site and vicinity. The CDR or framework region (e.g., an HC CDR3 described herein) may be, in certain embodiments, subjected to error-prone PCR to generate the diversity. This approach uses a “sloppy” version of PCR, in which the polymerase has a fairly high error rate (up to 2%), to amplify the wild-type sequence, and is generally described in Pritchard, et al. (2005) J. Theor. Biol. 234: 497-509 and Leung et al. (1989) Technique 1:11-15. Other exemplary mutagenesis techniques include DNA shuffling using random cleavage (Stemmer (1994) Nature 389-391; termed “nucleic acid shuffling”), RACHITT™ (Coco et al. (2001) Nature Biotech. 19:354), site-directed mutagenesis (Zoller et al. (1987) Nucl Acids Res 10:6487-6504), cassette mutagenesis (Reidhaar-Olson (1991) Methods Enzymol. 208:564-586) and incorporation of degenerate oligonucleotides (Griffiths et al. (1994) EMBO J. 13:3245).

In some embodiments of the invention, D segments in which a majority of the residues are either Ser or Tyr are picked. In some embodiments, when the DNA encoding the D region is synthesized, each Ser or Tyr residue is encoded by TMT, TMC, or TMY so that the encoded amino acid is either Ser or Tyr.

In some embodiments, the HC CDR3 sequences described herein may be subjected to selection for open reading frames by fusing the sequence encoding the HC CDR3 of interest in frame to an antibiotic resistance gene, such as Kan^(R) gene and selecting for kanamycin resistance. Cells in which the potential CDR3 has a stop codon or a frame shift will not have the antibiotic resistance and that sequence will be eliminated.

Methods of Construction of Libraries Comprising Human Antibody Heavy Chain CDR3s and Libraries Comprising Human Antibody Heavy Chain CDR3s

An antibody library is a collection of proteins that include proteins that have at least one immunoglobulin variable domain sequence. For example, camelized variable domains (e.g., VH domains) can be used as a scaffold for a library of proteins that include only one immunoglobulin variable domain sequence. In another example, the proteins include two variable domains sequences, e.g., a VH and VL domain, that are able to pair. An antibody library can be prepared from a nucleic acid library (an antibody-coding library) that includes antibody-coding sequences, e.g., comprising the sequences encoding the HC CDR3s provided herein.

In cases where a display library is used, each member of the antibody-coding library can be associated with the antibody that it encodes. In the case of phage display, the antibody protein is physically associated (directly or indirectly) with a phage coat protein. A typical antibody display library member displays a polypeptide that includes a VH domain and a VL domain. The display library member can display the antibody as a Fab fragment (e.g., using two polypeptide chains) or a single chain Fv (e.g., using a single polypeptide chain). Other formats can also be used.

As in the case of the Fab and other formats, the displayed antibody can include one or more constant regions as part of a light and/or heavy chain. In one embodiment, each chain includes one constant region, e.g., as in the case of a Fab. In other embodiments, additional constant regions are included. It is also possible to add one or more constant regions to a molecule after it is identified as having useful antigen binding site. See, e.g., US 2003-0224408.

Antibody libraries can be constructed by a number of processes (see, e.g., de Haard et al. (1999) J. Biol. Chem 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20, Hoogenboom et al. (2000) Immunol Today 21:371-8, and Hoet et al. (2005) Nat Biotechnol. 23(3):344-8.

In certain embodiments for constructing libraries, the heavy chains comprising the CDR3s described herein and the kappa and lambda light chains are best constructed in separate vectors. First, a synthetic gene is designed to embody each of the synthetic variable domains. The light chains may be bounded by restriction sites for ApaLI (positioned at the very end of the signal sequence) and AscI (positioned after the stop codon). The heavy chain may be bounded by SfiI (positioned within the Pe1B signal sequence) and NotI (positioned in the linker between CH1 and the anchor protein). Signal sequences other than Pe1B may also be used, e.g., a M13 pIII signal sequence.

The initial genes may be made with “stuffer” sequences in place of the desired CDRs. A “stuffer” is a sequence that is to be cut away and replaced by diverse DNA, but which does not allow expression of a functional antibody gene. For example, the stuffer may contain several stop codons and restriction sites that will not occur in the correct finished library vector. Stuffers are used to avoid have any one CDR sequence highly represented.

In another embodiment of the present invention, the heavy chain and the kappa or lambda light chains are constructed in a single vector or genetic packages (e.g., for display or display and expression) having appropriate restriction sites that allow cloning of these chains. The processes to construct such vectors are well known and widely used in the art. Preferably, a heavy chain and kappa light chain library and a heavy chain and lambda light chain library would be prepared separately.

Most preferably, the display is on the surface of a derivative of M13 phage. The most preferred vector contains all the genes of M13, an antibiotic resistance gene, and the display cassette. The preferred vector is provided with restriction sites that allow introduction and excision of members of the diverse family of genes, as cassettes. The preferred vector is stable against rearrangement under the growth conditions used to amplify phage.

In another embodiment of this invention, the diversity captured by the methods of the present invention may be displayed and/or expressed in a phagemid vector (e.g., pMID21 (DNA sequence shown in Table 35)) that displays and/or expresses the peptide, polypeptide or protein. Such vectors may also be used to store the diversity for subsequent display and/or expression using other vectors or phage.

In still other embodiments, a method termed the Rapid Optimization of LIght Chains or “ROLIC”, described in U.S. Ser. No. 61/028,265 filed Feb. 13, 2008, U.S. Ser. No. 61/043,938 filed Apr. 10, 2008, and U.S. Ser. No. 12/371,000 filed Feb. 13, 2009, a large population of LCs is placed in a phage vector that causes them to be displayed on phage. A small population (e.g., 3, 10, or 25) of HCs are cloned into E. coli so that the HCs are secreted into the periplasm, e.g., those HCs having the CDR3s described herein. The E. coli are then infected with the phage vectors encoding the large population of LCs to produce the HC/LC protein pairings on the phage. The phage particles carry only a LC gene.

In another aspect, in a method termed the Economical Selection of Heavy Chains or “ESCH”, also described in U.S. Ser. No. 61/028,265 filed Feb. 13, 2008, U.S. Ser. No. 61/043,938 filed Apr. 10, 2008, and U.S. Ser. No. 12/371,000 filed Feb. 13, 2009, a small population of LCs may be placed in a vector that causes them to be secreted. A new library of HCs in phage is constructed, such as those provided herein comprising the CDR3s. The LCs and HCs can then be combined by the much more efficient method of infection. Once a small set of effective HC are selected, these can be used as is, fed into ROLIC to obtain an optimal HC/LC pairing, or cloned into a Fab library of LCs for classical selection.

In another embodiment of this invention, the diversity captured by the methods of the present invention may be displayed and/or expressed using a vector suitable for expression in a eukaryotic cell, e.g., a yeast vector, e.g., for expression in a yeast cell.

Other types of protein display include cell-based display (see, e.g., WO 03/029,456); ribosome display (see, e.g., Mattheakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat Biotechnol. 18:1287-92); protein-nucleic acid fusions (see, e.g., U.S. Pat. No. 6,207,446); and immobilization to a non-biological tag (see, e.g., U.S. Pat. No. 5,874,214).

Antibodies isolated from the libraries of the present disclosure may be analyzed to determine the type of the LC and the closest germline gene. In a preferred embodiment, non-germline framework residues are changed back to the germline amino acid so long as binding affinity and specificity are not adversely affected to an unacceptable extent. The substitutions may be done as a group or singly. Human germline sequences are disclosed in Tomlinson, I. A. et al., 1992, J. Mol. Biol. 227:776-798; Cook, G. P. et al., 1995, Immunol. Today 16 (5): 237-242; Chothia, D. et al., 1992, J. Mol. Bio. 227:799-817. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, I. A. et al. MRC Centre for Protein Engineering, Cambridge, UK). Antibodies are “germlined” by reverting one or more non-germline amino acids in framework regions to corresponding germline amino acids of the antibody, so long as binding properties are substantially retained. Similar methods can also be used in the constant region, e.g., in constant immunoglobulin domains.

For example, an antibody can include one, two, three, or more amino acid substitutions, e.g., in a framework, CDR, or constant region, to make it more similar to a reference germline sequence. One exemplary germlining method can include identifying one or more germline sequences that are similar (e.g., most similar in a particular database) to the sequence of the isolated antibody. Mutations (at the amino acid level) are then made in the isolated antibody, either incrementally or in combination with other mutations. For example, a nucleic acid library that includes sequences encoding some or all possible germline mutations is made. The mutated antibodies are then evaluated, e.g., to identify an antibody that has one or more additional germline residues relative to the isolated antibody and that is still useful (e.g., has a functional activity). In one embodiment, as many germline residues are introduced into an isolated antibody as possible.

In one embodiment, mutagenesis is used to substitute or insert one or more germline residues into a framework and/or constant region. For example, a germline framework and/or constant region residue can be from a germline sequence that is similar (e.g., most similar) to the non-variable region being modified. After mutagenesis, activity (e.g., binding or other functional activity) of the antibody can be evaluated to determine if the germline residue or residues are tolerated (i.e., do not abrogate activity). Similar mutagenesis can be performed in the framework regions.

Selecting a germline sequence can be performed in different ways. For example, a germline sequence can be selected if it meets a predetermined criteria for selectivity or similarity, e.g., at least a certain percentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity. The selection can be performed using at least 2, 3, 5, or 10 germline sequences. In the case of CDR1 and CDR2, identifying a similar germline sequence can include selecting one such sequence. In the case of CDR3, identifying a similar germline sequence can include selecting one such sequence, but may include using two germline sequences that separately contribute to the amino-terminal portion and the carboxy-terminal portion. In other implementations, more than one or two germline sequences are used, e.g., to form a consensus sequence.

CDR1, CDR2, and Light-Chain Diversity

It is to be understood that the libraries of HC CDR3 are constructed in the background of diversity in HC CDR1, HC CDR2, and light chains. The light-chain diversity may be encoded in the same DNA molecule as the HC diversity or the LC and HC diversities may be encoded in separate DNA molecules. In Table 22 the fusion of a signal sequence::VH::CH1::His6::Myc::IIIstump (His6 is disclosed as SEQ ID NO: 934). CDR1 comprises residues 31-35; there is diversity at residues 31, 33, and 35. In one embodiment, residues 31, 33, and 35 can be any amino-acid type except cysteine. CDR2 comprises residues 50 through 65. There is diversity at positions 50, 52, 52a, 56, and 58. In one embodiment, residues 50, and 52 can be any of the types Ser, Gly, Val, Trp, Arg, Tyr; residue 52a can be Pro or Ser and residues 56 and 58 can be any amino-acid type except Cys. The diversity of HC CDR3 is cloned into a diversity of HC CDR1 and 2 that is at least 1. E 4, 1. E 5, 1. E 6, 1.E 7, 5. E 7, or 1. E 8.

In one embodiment, residues 31, 33, 35, 50, 52, 56, and 58 can be any amino-acid type except Cys or Met and residue 52a can be Gly, Ser, Pro, or Tyr. The diversity of HC CDR3 is cloned into a diversity of HC CDR1 and 2 that is at least 1. E 4, 1. E 5, 1. E 6, 1. E 7, 5. E 7, or 1. E 8.

In one embodiment, the diversity of the HC is cloned into a vector (phage or phagemid) that contains a diversity of light chains. This diversity is at least 25, 50, 100, 500, 1. E 3, 1. E 4, 1. E 5, 1. E 6, or 1. E7. The diversity of HC CDR3 is at least 221, 272, 500, 1000, 1. E4, 1. E 5, 1. E 6, 1.E7, 1. E 8, or 1. E 9.

In one embodiment, the diversity of the HC is cloned into a phage vector that displays the HC on a phage protein such as III, VIII, VII, VI, or IX or a fragment of one of these sufficient to cause display and light chains are combined with the HC by infecting a cell collection wherein each cell secrets a light chain. The diversity of the light chains in the cells is at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, or 100. The diversity of HC CDR3 is at least 221, 272, 500, 1000, 1. E 4, 1.E 5, 1.E 6, 1. E7, 1. E 8, or 1. E 9.

Table 30 shows the sequence of the phage vector DY3FHC87 (SEQ ID NO:894) which carries a bla gene, a display cassette for heavy chains under control of a Plac promoter. DY3FHC87 contains all the genes of M13 as well. Infecting F+ E. coli cells that harbor a diversity of light chains in a vector such as pLCSK23 (Sequence in Table 40) (SEQ ID NO:896). The vector pLCSK23 carries a Kan^(R) gene. Under the control of Plac promoter, there is a gene beginning at base 2215 having a signal sequence (bases 2215-2277), a VL (in this sequence the VL encodes the sequence shown in (SEQ ID NO:897) from base 2278 to base 2598, Ckappa from base 2599 to 2922, a linker that allows an NotI site from 2923 to 2931, and a V5 tag (bases 2932-2973). There are an SfiI site at 2259-2271 and a KpnI site at 2602-2605 to allow easy replacement of Vkappas. (SEQ ID NO:897) is an example of the proteins that are secreted. It is to be understood that CKappa and the V5 tag are constant. All of the proteins shown in Table 19 (VK1O2gl-JK3, VK1O2var1, VK1O2var2, VK1O2var3, VK1O2var4, VK1O2var5, VK3L6gl-JK4, VK3L6var1, VK3L6var2, VK3L6var3, VK3L6var4, VK3L6var5, VK3L6var6, VK3L6var7, VK3L6var8, VK3A27gl-JK3, VK3A27var1, VK3A27var2, VK3A27var3, VK3A27var4, VK3A27var5, VK3A27var6, VK3A27var7, VK3L2gl-JK3, and VK1glL8-JK5) will have these sequences attached at the carboxy end.

Light Chain Diversity

Table 800 shows a kappa LC (light chain) that is known to pair well with 3-23 and with five CDR mutations with one HC based on 3-23, LC K1(O12)::JK1 makes a high affinity Ab to a protein target. O12 is a frequently used VKI. The gene has been designed to have useful, distinct restriction sites in the signal sequence (ApaLI), FR1 (XhoI, SgfI), FR2 (KpnI), FR3(XbaI), and Fr4::Ckappa (BsiWI) so that each CDR and be replaced with a varied population.

In human LCs, CDR3 is most important and CDR1 is next most important. CDR2 seldom makes contact with the Ag. Diversity is introduced into the CDRs as shown in Table 900 and Table 1000 (CDR1), Table 1100 and Table 1200 (CDR2), Tables 1300, 1400, and 1500 (CDR3). For Economical Selection of Heavy Chains (ESHC), a small number, for example, 50 LCs with diversity in CDR3 as in Table 1200 are picked for expression in pLCSK24 for secretion into the periplasm. More LCs can be used if several cell lines are maintained so that each cell line contains, for example, 50 or fewer LC.

Table 900 shows diversity for LC CDR1. The library can contain the O12 residue with the added diversity of the AA types shown as “allowed”; reading “allowed” as “additional allowed types” in Tables 900, 1000, 1100, 1200, 1300, 1400. O12 has R₂₄ASQSISSYLN₃₄ (SEQ ID NO: 935). Other VK1 loci have Q at 24. Other loci have M at 25. S₂₆ and Q₂₇ are invariant in VKI. Other VKI loci have D or G at 28. I₂₉ and L₃₃ are invariant in VKI and the side groups are oriented inward. Other VKI loci allow the diversity shown in Table 900 at positions 30, 31, 32, and 34. In Table 900, only seven of the eleven positions are varied and the total diversity is 576.

Table 1000 shows a higher level of diversity for LC CDR1. Here 8 of 11 positions have been varied. Those that are constant are either far from the combining site or have buried side groups.

Table 1100 shows a low level variegation for CDR2. CDR2 is far from the antigen combining site and diversity here may not be very useful. Indeed, the GL diversity is very limited. Table 1100 includes the GL diversity. Table 1200 contains a higher level of diversity, 1920 sequences allowed.

Table 1300 shows a low level of diversity for LC CDR3, 2160 sequences. Table 1400 shows a higher level which allows 105,840 sequences.

For ROLIC, about 3×10⁷ LC are produced having the diversity shown in Tables 900, 1100, and 1300.

Heavy Chain Diversity

Ab HC (heavy chain) have diversity in CDR1, CDR2, and CDR3. The diversity in CDR3 is especially complex because there is both sequence and length diversity. The sequence diversity is not random. Cells making Ab genes join a V segment to a D segment to a JH segment. The D segment is optional; about half of natural human Abs have a recognizable D. There can be extensive editing at the V-D, D-J, or V-J boundaries with none to many bases added or removed. An Ab that has a germline V::D::JH could be viewed as a germline Ab.

Human D segments are shown in Table 21. Each germline (GL) D segment may appear in an Ab gene in any of the three forward reading frames. In some reading frames, some of the D segments encode stop codons. These D segments do occur rarely with the stop codon modified. Table 600 shows the frequency of each D segment as a percent of all observed D segments. Most of the examples herein that contain D segments use Ds that are fairly common (>2% of all observed Ds).

In one aspect, the present invention involves composing Ab HC genes by fusing 3-23 (or another VH, such as 4-34) to one of a) a number of amino acids picked from the set comprising (S, Y, D, R, N), b) a D region, c) a JH region, and d) the FR4 portion of a JH region. These fusions can be a GL 3-23 or a 3-23 that has synthetic diversity in CDR1 and/or CDR2. The lengths of the HC CDR3 and be any number from about 3 to about 24. Preferably, the library would contain member with HC CDR3 of lengths 6, 8, 10, 12, 14, 16, 18, and 20. Alternatively, the lengths could be 5, 8, 11, 14, 17, and 20 or any other combination.

Table 21 shows a number of examples of designs of suitable CDR3s with lengths from 6 to 20. The codons that specify the uppercase letters in column 2 are to be synthesized with wobbling. Column 3 shows the level of doping. Table 100 shows ratios in which the various lengths of HC CDR3 could be combined to form a library that is expected to contain Abs that bind almost all protein targets.

TABLE 100 Length 6 8 10 12 14 16 20 Diversity 1. × 2. × 4. × 10⁵ 8. × 10⁵ 8. × 10⁵ 8. × 10⁵ 4. × 10⁵ 10⁵ 10⁵

For length 6, Table 21 four examples are given. For example, 6a has VH(3-23) joined directly to JH1 with the first six AAs wobbled, 6b has Tyr joined to D4-17 in second reading frame joined to the FR4 AAs of JH1, and 6c has D5-5(3) joined to the FR residues of JH1. Since these give different kinds of diversity, including all is preferred, but a library containing only one of these should give useful Abs.

For length 8, Table 21 shows three examples. 8a has YY fused to all of JH1 while 8b has one Y fused to D6-13(1) fused to the FR region of JH1. Lengths 10, 12, 14, 16, and 20 are also shown in Table 21. The HC CDR3 diversity could be built in a germline 3-23 or 3-23 containing synthetic diversity. Alternatively, a different VH, such as 4-34 could be used.

ROLIC is a method in which a small population of HCs are expressed in F⁺ E. coli as soluble proteins. The population is infected with phage that carry LC::III_(stump) fusions. The phage produced obtain a HC from the periplasm of the cell that produces them. These phage can be bound to immobilized target and the binder are separated from the non-binders. The size of the population is important because when the recovered phage are propagated, the recovered phage must find the same type of cell as it came from to continue the association between LC and HC. Thus it is desirable that the number of HC be small in each cell line. Thus it may be desirable to maintain a number of cell lines with up to 10, 20, 30, or 40 different HC in each cell line. Thus we may have 1, 2, 4, 6, 8, 10, 24, 48, or 96 cell lines and we perform the same number of parallel phage productions, selections, and amplifications. After one or two rounds, we test colonies for production of phage that bind the target by an ELISA assay. Each ELISA colony contains a useful LC and a useful HC, but they are not on the same piece of DNA. Nevertheless, we know the start and end of each LC and each HC and can therefore use PCR on the colony to produce a Fab display or Fab secretion cassette that can be put into a display phage or phagemid or into a Fab-production plasmid.

In Efficient Selection of HCs (ESHC), we reverse the roles of LC and HC in ROLIC and have LCs in a plasmid so that they are produced as soluble proteins in the periplasm of F⁺ E. coli. We produce the HC diversity in a phage vector that has no LC gene. We infect the LC-producing F⁺ E. coli with the HC-carrying phage. We obtain phage that carry an HC gene and both HC and LC proteins. We select these phage for binding to the target. In many Abs, the LC is permissive and does not contribute greatly to binding affinity. Picking the best LC can greatly increase affinity, but it is usually possible to select a Fab with a very limited repertoire of LCs. Thus, we place a small set of LCs, preferable germline in the framework regions in the LC-producing F⁺ E. coli. If there are, for example, 25 LC in the LC cell line, then we obtain a 25-fold reduction in the number of cell transformants that need to be made.

The libraries described have a range of HC CDR3 lengths. To favor proper folding, the HC CDR3 have either a D segment or no D segment joined to most, all, or the framework portion of a JH segment. The sequences are diversified by using wobble DNA synthesis. Although this theoretically allows any amino-acid type at any position, in practice, the actual sequences are strongly biased toward the parental sequences and AA types that are close in the genetic code table.

By using ESHC, we can sample new designs of synthetic HC CDR3 diversity. In the examples given, we use a pool of, for example, 50 LCs. A library of 5×10⁸ HC should perform as well as an old-style library of 2.5×10¹⁰ but require far less effort.

When wobbling a sequence, picking the initial codons affects the actual mixture of AAs seen in the library. Table 300 shows which amino-acid substitutions require 1, 2, or 3 base changes from each starting parental codon. For example, if we start with get or gcc for Ala, all three stop codons require three base changes and so are rare. If using 76:8:8:8 mixtures, Ala will appear in 57% of the cases (0.76*0.76). V, G, T, P, S will each appear in about 6% and D about 3%. E, I, L, F, Y, H, N, C, and R will be down about 10-fold. M, W, Q, K, Am, Oc, and Op will be even rarer. If we started with gca, then E would replace D in needing only one base change, but opal and ochre stops require only two base changes, which is undesirable. The preferred codons are marked with a star (*). The choice for serine is complicate our desire to have Y substitute for S with high frequency. This brings Op and Oc into the group that differ from the parent by only two bases. This problem can be overcome by cloning the HC CDR3 repertoire before an antibiotic resistance gene such as KanR or AmpR and selecting for resistance, thus eliminating the members that contain stop codons. In addition, the library can be produced in supE E. coli which insert Q instead of stopping.

TABLE 300 Amino Parental acid codon 1 base change 2 base changes 3 base changes A* gct, gcc V, D, G, T, P, S E, I, L, F, Y, H, N, C, R M, W, Q, K, Am, Oc, Op A gca V, E, G, T, P, S D, I, L, Oc, Q, K, Op, R M, W, H, N, C, Am, F, Y A gcg V, E, G, T, P, S D, M, L, Am, Q, K, R, W I, F, Y, Oc, Op, H, N, C C tgt, tgc Y, S, F, W, Op, L, H, N, D, P, T, A, V, I Am, Oc, Q, K, E, M R, G D gat, gac E, G, A, V, N, F, S, C, L, P, Q, K, R, Oc, M, W, Op H, Y Am, I, T E gaa D, G, A, V, K, Am, L, I, S, P, T, R, Op, Y, M, F, C, W Q, Oc H, N E* gag D, G, A, V, K, M, L, S, P, T, Y, H, N, Oc, F, C, I, Op Q, Am R, W F ttt, ttc L, I, V, S, Y, C M, Am, Op, Oc, W, P, T, Q, K, E A, H, N, D, R, G G* ggt, ggc D, A, V, S, R, C E, W, F, L, I, T, P, Y, H, N Am, Oc, Op, M, Q, K G gga E, A, V, R, Oc D, W, L, I, S, P, T, Op, Q, Am, Oc, M, F, Y, H, N K G ggg E, A, V, R, W D, Oc, L, M, S, P, T, Am, Oc, I, F, Y, H, N Op, Q, K H cat, cac Q, Y, N, D, L, F, S, C, I, T, V, A, D, G, Op, W, M, E P, R Am, Oc I* att, atc M, L, F, V, T, Y, C, P, H, R, A, D, G Am, Op, Oc, W, Q, K, E N, S I ata M, L, V, T, K, R Op, Oc, S, P, Q, A, E, G, F, Am, C, D, H, W, Y N K aaa N, Q, Oc, E, P, H, Y, D, M, L, V, S, T, A, C, F, W I, R Am, Op, G K* aag N, Q, Am, E, P, H, Y, D, I, L, V, S, T, A, C, F, Op M, R Oc, G, W L tta F, S, Oc, Op, I, Y, C, W, M, P, T, A, Q, K, D, H, N V E, R, G, Am L ttg F, S, Am, W, M, Y, C, Oc, Op, P, T, A, Q, D, H, N V K, E, R, G, I L* ctt, ctc F, I, V, P, H, R M, S, Y, C, T, N, A, D, G Am, Oc, Op, W, E, K, Q L cta I, V, P, Q, R F, M, S, Oc, Op, T, K, A, E, Am, W, D, N, C, Y G, H L ctg M, V, P, Q, R F, I, S, Am, T, K, A, E, G, Oc, Op, D, N, C, Y H, W M atg L, V, T, K, R, I F, N, S, P, A, Am, Q, E, W, Oc, Op, Y, C, H, D G N aat, aac K, Y, H, D, I, T, F, C, L, P, R, V, A, G, M, Op, W S Q, E, Am, Oc P* cct, ccc S, T, A, L, H, R F, Y, C, I, N, V, D, G, Q Am, Oc, Op, W, M, E, K P cca S, T, A, L, Q, R Oc, Op, I, K, V, E, G, H Am, W, M, D, N, C, F, Y P ccg S, T, A, L, Q, R Am, M, K, V, E, G, H C, D, F, I, N, W, Y, Oc, Op Q caa Oc, K, E, R, P, Y, Am, N, D, S, T, A, I, V, F, C, W, M L, H G, Op Q* cag H, Am, K, E, R, N, D, Y, M, T, V, A, G, W, C, F, Op, I P, L Oc, S R* cgt, cgc C, S, G, H, P, L Op, W, Q, F, Y, I, T, N, V, Am, Oc, M, E, K A, D R cga G, Op, Q, P, L Oc, S, C, W, H, I, V, T, A, Am, M, C, D, N, F, Y E, K R cgg G, W, Q, P, L Am, Op, S, M, V, T, A, K, F, Y, I, Oc, D, N E, H, C R aga G, Op, S, K, T, I C, W, N, M, L, V, P, A, Oc, F, Y, H, D, Am Q, E R agg G, W, S, K, T, C, Op, Am, L, I, V, A, Q, F, Y, H, D, Oc M P, E, N S* tct, tcc F, Y, C, P, T, A L, Oc, Op, Am, W, I, V, N, E, K, M, Q D, R, G, H S tca L, Oc, Op, P, T, F, Y, C, W, Q, R, I, K, V, E, M, W, D, N, H A G, Am S tcg L, Am, W, P, T, F, Y, C, Op, Oc, Q, R, M, I, D, N, H A K, V, E, G S agt, agc C, R, G, N, T, I F, Y, L, P, H, V, A, D, K, Am, Oc, M, E, Q W, Op T* act, acc S, P, A, I, N F, Y, C, L, H, R, M, K, V, Am, Oc, Op, W, E, Q D, G T aca S, P, A, I, K, R L, Oc, Op, Q, M, E, G, V, F, Y, C, Am, W, D, H N T acg S, P, A, M, K, R I, N, L, Am, W, Q, V, E, G C, F, Y, Oc, Op, D, H V* gtt, gtc F, L, I, A, D, G S, P, T, Y, H, N, E, C, R, M Am, Oc, Op, W, Q, K V gta L, I, A, E, G F, M, D, S, P, T, Oc, Op, Q, Am, W, C, Y, H, N R, K V gtg L, M, A, E, G F, I, D, S, P, T, Am, Q, R, Oc, Op, C, Y, H, N K, W W tgg C, R, G, Am, S, P, Q, F, M, T, K, V, A, E, D, N, H, I L, Op Oc, Y Y tat, tac C, S, F, N, H, L, W, Q, K, E, P, I, T, V, A, M D, Oc, Am G, Op, R Am is TAG stop, Op is TGA, Oc is TAA

Methods of Using the Libraries

Off-Rate Selection.

Since a slow dissociation rate can be predictive of high affinity, particularly with respect to interactions between polypeptides and their targets, the methods described herein can be used to isolate ligands with a desired kinetic dissociation rate (i.e., reduced) for a binding interaction to a target.

To select for slow dissociating antibodies from a display library, the library is contacted to an immobilized target. The immobilized target is then washed with a first solution that removes non-specifically or weakly bound antibodies. Then the bound antibodies are eluted with a second solution that includes a saturating amount of free target, i.e., replicates of the target that are not attached to the particle. The free target binds to antibodies that dissociate from the target. Rebinding of the eluted antibodies is effectively prevented by the saturating amount of free target relative to the much lower concentration of immobilized target.

The second solution can have solution conditions that are substantially physiological or that are stringent (e.g., low pH, high pH, or high salt). Typically, the solution conditions of the second solution are identical to the solution conditions of the first solution.

Fractions of the second solution are collected in temporal order to distinguish early from late fractions. Later fractions include antibodies that dissociate at a slower rate from the target than biomolecules in the early fractions. Further, it is also possible to recover antibodies that remain bound to the target even after extended incubation. These can either be dissociated using chaotropic conditions or can be amplified while attached to the target. For example, phage bound to the target can be contacted to bacterial cells.

Selecting or Screening for Specificity.

The display library screening methods described herein can include a selection or screening process that discards antibodies that bind to a non-target molecule. Examples of non-target molecules include, e.g., a carbohydrate molecule that differs structurally from the target molecule, e.g., a carbohydrate molecule that has a different biological property from the target molecule. In the case of a sulfated carbohydrate, a non-target may be the same carbohydrate without the sulfate or with the sulfate in a different position. In the case of a phosphopeptide, the non-target may be the same peptide without the phosphate or a different phosphopeptide.

In one implementation, a so-called “negative selection” step is used to discriminate between the target and related non-target molecule and a related, but distinct non-target molecules. The display library or a pool thereof is contacted to the non-target molecule. Members that do not bind the non-target are collected and used in subsequent selections for binding to the target molecule or even for subsequent negative selections. The negative selection step can be prior to or after selecting library members that bind to the target molecule.

In another implementation, a screening step is used. After display library members are isolated for binding to the target molecule, each isolated library member is tested for its ability to bind to a non-target molecule (e.g., a non-target listed above). For example, a high-throughput ELISA screen can be used to obtain this data. The ELISA screen can also be used to obtain quantitative data for binding of each library member to the target. The non-target and target binding data are compared (e.g., using a computer and software) to identify library members that specifically bind to the target.

In certain embodiments, the antibodies comprising the CDR3s of the invention may be able to bind carbohydrates. Methods for evaluating antibodies for carbohydrate binding include ELISA, immunohistochemistry, immunoblotting, and fluorescence-activated cell sorting. These methods can be used to identify antibodies which have a K_(D) of better than a threshold, e.g., better than 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 100 pM, or 10 pM.

ELISA.

Proteins encoded by a display library can also be screened for a binding property using an ELISA assay. For example, each protein is contacted to a microtitre plate whose bottom surface has been coated with the target, e.g., a limiting amount of the target. The plate is washed with buffer to remove non-specifically bound polypeptides. Then the amount of the protein bound to the plate is determined by probing the plate with an antibody that can recognize the polypeptide, e.g., a tag or constant portion of the polypeptide. The antibody is linked to an enzyme such as alkaline phosphatase, which produces a calorimetric product when appropriate substrates are provided. The protein can be purified from cells or assayed in a display library format, e.g., as a fusion to a filamentous bacteriophage coat. Alternatively, cells (e.g., live or fixed) that express the target molecule, e.g., a target that contains a carbohydrate moiety, can be plated in a microtitre plate and used to test the affinity of the peptides/antibodies present in the display library or obtained by selection from the display library.

In another version of the ELISA assay, each polypeptide of a diversity strand library is used to coat a different well of a microtitre plate. The ELISA then proceeds using a constant target molecule to query each well.

Cell Binding Assays.

Antibodies can be evaluated for their ability to interact with one or more cell types, e.g., a hematopoietic cell. Fluorescent activated cell sorting (FACS) is one exemplary method for testing an interaction between a protein and a cell. The antibody is labeled directly or indirectly with a fluorophore, before or after, binding to the cells, and then cells are counted in a FACS sorter.

Other cell types can be prepared for FACS by methods known in the art.

Homogeneous Binding Assays.

The binding interaction of candidate polypeptide with a target can be analyzed using a homogenous assay, i.e., after all components of the assay are added, additional fluid manipulations are not required. For example, fluorescence resonance energy transfer (FRET) can be used as a homogenous assay (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first molecule (e.g., the molecule identified in the fraction) is selected such that its emitted fluorescent energy can be absorbed by a fluorescent label on a second molecule (e.g., the target) if the second molecule is in proximity to the first molecule. The fluorescent label on the second molecule fluoresces when it absorbs to the transferred energy. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay should be maximal. A binding event that is configured for monitoring by FRET can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter). By titrating the amount of the first or second binding molecule, a binding curve can be generated to estimate the equilibrium binding constant.

Another example of a homogenous assay is Alpha Screen (Packard Bioscience, Meriden Conn.). Alpha Screen uses two labeled beads. One bead generates singlet oxygen when excited by a laser. The other bead generates a light signal when singlet oxygen diffuses from the first bead and collides with it. The signal is only generated when the two beads are in proximity. One bead can be attached to the display library member, the other to the target. Signals are measured to determine the extent of binding.

The homogenous assays can be performed while the candidate polypeptide is attached to the display library vehicle, e.g., a bacteriophage.

Surface Plasmon Resonance (SPR).

The binding interaction of a molecule isolated from a display library and a target can be analyzed using SPR. SPR or Biomolecular Interaction Analysis (BIA) detects biospecific interactions in real time, without labeling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The changes in the refractivity generate a detectable signal, which are measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in U.S. Pat. No. 5,641,640; Raether (1988) Surface Plasmons Springer Verlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705 and on-line resources provide by BIAcore International AB (Uppsala, Sweden).

Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (K_(D)), and kinetic parameters, including k_(on) and k_(off), for the binding of a biomolecule to a target. Such data can be used to compare different biomolecules. For example, proteins encoded by nucleic acid selected from a library of diversity strands can be compared to identify individuals that have high affinity for the target or that have a slow k_(off). This information can also be used to develop structure-activity relationships (SAR). For example, the kinetic and equilibrium binding parameters of matured versions of a parent protein can be compared to the parameters of the parent protein. Variant amino acids at given positions can be identified that correlate with particular binding parameters, e.g., high affinity and slow k_(off). This information can be combined with structural modeling (e.g., using homology modeling, energy minimization, or structure determination by crystallography or NMR). As a result, an understanding of the physical interaction between the protein and its target can be formulated and used to guide other design processes.

Protein Arrays.

Proteins identified from the display library can be immobilized on a solid support, for example, on a bead or an array. For a protein array, each of the polypeptides is immobilized at a unique address on a support. Typically, the address is a two-dimensional address. Methods of producing polypeptide arrays are described, e.g., in De Wildt et al. (2000) Nat. Biotechnol. 18:989-994; Lueking et al. (1999) Anal. Biochem. 270:103-111; Ge (2000) Nucleic Acids Res. 28, e3, I-VII; MacBeath and Schreiber (2000) Science 289:1760-1763; WO 01/40803 and WO 99/51773A1. Polypeptides for the array can be spotted at high speed, e.g., using commercially available robotic apparati, e.g., from Genetic MicroSystems or BioRobotics. The array substrate can be, for example, nitrocellulose, plastic, glass, e.g., surface-modified glass. The array can also include a porous matrix, e.g., acrylamide, agarose, or another polymer.

Kits

Also provided are kits for use in carrying out a method according to any aspect of the invention. The kits may include the necessary vectors. One such vector will typically have an origin of replication for single stranded bacteriophage and either contain the sbp member nucleic acid or have a restriction site for its insertion in the 5′ end region of the mature coding sequence of a phage capsid protein, and with a secretory leader coding sequence upstream of said site which directs a fusion of the capsid protein exogenous polypeptide to the periplasmic space.

Also provided are packages encoding the HC CDR3s as defined above and polypeptides comprising the HC CDR3s and fragments and derivatives thereof, obtainable by use of any of the above defined methods. The derivatives may comprise polypeptides fused to another molecule such as an enzyme or a Fc tail.

The kit may include a phage vector (e.g., DY3F87HC) which has a site for insertion of HC CDR3s for expression of the encoded polypeptide in free form. The kit may also include a plasmid vector for expression of soluble light chains, e.g., pLCSK23. The kit may also include a suitable cell line (e.g., TG1). The diversity of light chains encoded by pLCSK23 may be 10, 15, 20, 25, 30, or 50. The LCs in the diversity may be constructed or picked to have certain desirable properties, such as, being germline in the framework regions and having diversity in CDR3 and/or CDR1. The germlines may be of highly utilized ones, e.g., VK1_2-02, VK3_1-A27, VK3_5-L6, VK3_3-L2 for kappa and VL2_2a2, VL1_1c, VL1_1g, VL3_3r for lambda.

For example, one could clone genes for

VK1O2gl-JK3, VK1O2var1, VK1O2var2, VK1O2var3, VK1O2var4, VK1O2var5, VK3L6gl-JK4, VK3L6var1, VK3L6var2, VK3L6var3, VK3L6var4, VK3L6var5, VK3L6var6, VK3L6var7, VK3L6var8, VK3A27gl-JK3, VK3A27var1, VK3A27var2, VK3A27var3, VK3A27var4, VK3A27var5, VK3A27var6, VK3A27var7, VK3L2gl-JK3, VK1glL8-JK5, and VK1GLO12-JK3 (amino-acid sequences shown in Table 19) into pLCSK23.

TABLE 19 26 VL to be used in pLCSK23. VK1O2gl-JK3 (SEQ ID NO: 4) DIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPFTFGP GTKVDIK 107 VK1O2var1 (SEQ ID NO: 5) S28D DIQMTQSPSS LSASVGDRVT ITCRASQDIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPFTFGP GTKVDIK 107 VK1O2var2 (SEQ ID NO: 6) S91R DIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ RYSTPFTFGP GTKVDIK 107 VK1O2var3 (SEQ ID NO: 7) S91E DIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ EYSTPFTFGP GTKVDIK 107 VK1O2var4 (SEQ ID NO: 8) S31R DIQMTQSPSS LSASVGDRVT ITCRASQSIS RYLNWYQQKP GKAPKLLIYA ASSLQSGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPFTFGP GTKVDIK 107 VK1O2var5 (SEQ ID NO: 9) S31E, S93R DIQMTQSPSS LSASVGDRVT ITCRASQSIS EYLNWYQQKP GKAPKLLIYA ASSLQSGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYRTPFTFGP GTKVDIK 107 VK3L6gl-JK4 (SEQ ID NO: 10) EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGG GTKVEIK 107 VK3L6var1 (SEQ ID NO: 11) S31R EIVLTQSPAT LSLSPGERAT LSCRASQSVS RYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGG GTKVEIK 107 VK3L6var2 (SEQ ID NO: 12) S92R EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RRNWPLTFGG GTKVEIK 107 VK3L6var3 (SEQ ID NO: 13) S92G EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RGNWPLTFGG GTKVEIK 107 VK3L6var4 (SEQ ID NO: 14) S92Y EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RYNWPLTFGG GTKVEIK 107 VK3L6var5 (SEQ ID NO: 15) S92E EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RENWPLTFGG GTKVEIK 107 VK3L6var6 (SEQ ID NO: 16) Y32F EIVLTQSPAT LSLSPGERAT LSCRASQSVS SFLAWYQQKP GQAPRLLIYD ASNRATGIPA 60 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGG GTKVEIK 107 VK3L6var7 (SEQ ID NO: 17) Y32D EIVLTQSPAT LSLSPGERAT LSCRASQSVS SDLAWYQQKP GQAPRLLIYD ASNRATGIPA 60 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGG GTKVEIK 107 VK3L6var8 (SEQ ID NO: 18) N93G EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSGWPLTFGG GTKVEIK 107 VK3A27gl-JK3 (SEQ ID NO: 19) EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP 60 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPFTFG PGTKVDIK 108 VK3A27var1 (SEQ ID NO: 20) S31R EIVLTQSPGT LSLSPGERAT LSCRASQSVS RSYLAWYQQK PGQAPRLLIY GASSRATGIP 60 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPFTFG PGTKVDIK 108 VK3A27var2 (SEQ ID NO: 21) S32R EIVLTQSPGT LSLSPGERAT LSCRASQSVS SRYLAWYQQK PGQAPRLLIY GASSRATGIP 60 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPFTFG PGTKVDIK 108 VK3A27var3 (SEQ ID NO: 22) S32D EIVLTQSPGT LSLSPGERAT LSCRASQSVS SDYLAWYQQK PGQAPRLLIY GASSRATGIP 60 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPFTFG PGTKVDIK 108 VK3A27var4 (SEQ ID NO: 23) G93E EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP 60 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYESSPFTFG PGTKVDIK 108 VK3A27var5 (SEQ ID NO: 24) G93R EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP 60 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYRSSPFTFG PGTKVDIK 108 VK3A27var6 (SEQ ID NO: 25) S30D, G93E EIVLTQSPGT LSLSPGERAT LSCRASQSVD SSYLAWYQQK PGQAPRLLIY GASSRATGIP 60 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYESSPFTFG PGTKVDIK 108 VK3A27var7 (SEQ ID NO: 26) S94R EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGIP 60 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGRSPFTFG PGTKVDIK 108 VK3L2gl-JK3 (SEQ ID NO: 27) EIVMTQSPAT LSVSPGERAT LSCRASQSVS SNLAWYQQKP GQAPRLLIYG ASTRATGIPA 60 RFSGSGSGTE FTLTISSLQS EDFAVYYCQQ YNNWPFTFGP GTKVDIK 107 VK1glL8-JK5 (SEQ ID NO: 28) DIQLTQSPSF LSASVGDRVT ITCRASQGIS SYLAWYQQKP GKAPKLLIYA ASTLQSGVPS 60 RFSGSGSGTE FTLTISSLQP EDFATYYCQQ LNSYPITFGQ GTRLEIK 107 VK1GLO12-JK3 (SEQ ID NO: 897) DIQMTQSPSS LSASVGDRV TITCRASQSI SSYLNWYQQK PGKAPKLLIY AASSLQSGVP 60 SRFSGSGSGT DFTLTISSL QPEDFATYYC QQSYSTPFTF GPGTKVDIKR GTVAAPSVFI 120 FPPSDEQLKS GTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS 180 STLTLSKADY EKHKVYACE VTHQGLSSPV TKSFNRGECA AAGKPIPNPL LGLDST 236

The kits may include ancillary components required for carrying out the method, the nature of such components depending of course on the particular method employed. Useful ancillary components may comprise helper phage, PCR primers, buffers, and/or enzymes of various kinds. Buffers and enzymes are typically used to enable preparation of nucleotide sequences encoding Fv, scFv or Fab fragments derived from rearranged or unrearranged immunoglobulin genes according to the strategies described herein.

Methods of Introducing Diversity

There are many ways of generating DNA that is variable. One way is to use mixed-nucleotide synthesis (MNS). One version of MNS uses equimolar mixtures of nucleotides as shown in Table 5. For example, using NNK codons gives all twenty amino acids and one TAG stop codon. The distribution is 3(R/S/L): 2(A/G/V/T/P): 1(C/D/E/F/H/I/K/M/N/Q/W/Y) (e.g., 3 of each of Arg, Ser, and Leu, and so forth). An alternative, herein termed “wobbling”, uses mixed nucleotides but not in equimolar amounts. For example, if a parental codon were TTC (encoding Phe), we could use a mixture of (0.082 T, 0.06 C, 0.06 A, and 0.06 G) in place of T and a mixture of (0.082 C, 0.06 T, 0.06 A, and 0.06 G) in place of C. This would give TTC or TTT (encoding Phe) 59% of the time and Leu 13%, S/V/I/C/Y ˜5%, and other amino-acid types less often.

Van den Brulle et al. (Biotechniques 45:340-3 (2008)) describe a method of synthesis of variable DNA in which type IIs restriction enzymes are used to transfer trinucleotides from an anchored hair-pin oligonucleotide (PHONs) to a so called “splinker”. See also EP patents 1 181 395, EP 1 411 122, EP 1 314 783 and EP applications EP 01127864.5, EP 04001462.3, EP 08006472.8. By using mixtures of anchored PHONs and splinkers, one can build libraries in which desired amino-acid types are allowed in designer-determined ratios. Thus, one can direct that one amino-acid type is present, for example 82% of the time and 18 other amino-acid types (all non-parental amino-acid types except Cys) are present at 2% each. Herein, we will refer to such a synthesis as “dobbling” (digital wobbling). In some aspects, dobbling is preferred to wobbling, but wobbling provides useful embodiments, partly because the structure of the genetic code table causes wobbling to make mostly conservative substitutions. Dobbling does offer the possibility to exclude unwanted amino-acid types. In CDRs, unpaired cysteines are known, even in Abs approved as therapeutics, but in some embodiments, one would like to avoid them. In some embodiments, when diversifying a D region that contains a pair of cysteines, the cysteins are not allowed to vary because the disulfide-closed loop is an important structural element and because one does not want unpaired cysteines.

In addition, one can synthesize a DNA molecule that encodes a parental amino-acid sequence and subject that DNA to error-prone PCR using primers that cover the framework regions so that mutations in the framework regions are avoided.

TABLE 5 Standard codes for mixed nucleotides N is equimolar A, C, G, T B is equimolar C, G, T (not A) D is equimolar A, G, T (not C) H is equimolar A, C, T (not G) V is equimolar A, C, G (not T) K is equimolar G, T (Keto) M is equimolar A, C (aMino) R is equimolar A, G (puRine) S is equimolar C, G (Strong) W is equimolar A, T (weak) Y is equimolar C, T (pYrimidine)

TABLE 6 Example of mixed nucleotides for wobbling e = 0.82 A + 0.06 C + 0.06 G + 0.06 T q = 0.06 A + 0.82 C + 0.06 G + 0.06 T j = 0.06 A + 0.06 C + 0.82 G + 0.06 T z = 0.06 A + 0.06 C + 0.06 G + 0.82 T

EXEMPLIFICATION

The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.

Prophetic Example 1: Libraries with Very Short HC CDR3s

Very short HC CDR3s have been described in the art. Kadirvelraj et al. (2006) Proc. Natl. Acad. Sci. USA 103:8149-54 have described a four amino-acid HC CDR3 sequence in an antibody that binds Streptococcus Type B III Ag (GBS-Ag) but not to Streptococcus pneumoniae capsular Ag. GBS-Ag is sialylated at regular intervals. S. pneumoniae capsular Ag (SPC-Ag) is very similar but lacks the sialic acid groups. Such a short HC CDR3 creates a wide groove into which a carbohydrate could bind, and such Abs are very, very rare in existing antibody libraries. Thus, current libraries do not afford a large variety of potential binders to carbohydrates.

Ab 1B1 is the murine mAb that binds GBS-Ag; Ab 1QFU is the mAb having a known 3D structure and the closest sequence; and 1NSN is an antibody of known 3D structure having a HC CDR3 of length 4. Examination of a 3-23 HC structure gives a distance from Cα of R₉₄ (which ends FR3) to the Cα of the W₁₀₄ (which begins FR4) of ˜10 Å. The CDR3 of 1B1 (NWDY (SEQ ID NO:29)) shows that the AAs need not have only small side groups or be mostly of glycine. Three amino acids (AAs) can bridge 10 Å, although PPP might not work. Indeed, we have obtained a few Fabs with CDR3s as short as 3 AAs, but they are very rare.

Although short and very short HC CDR3s have been described, no one has suggested making an Ab library having many members (e.g., greater than about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of members) with short HC CDR3s (e.g., HC CDR3s of 3 to 5 amino acids). One approach to building an effective library is to first design amino-acid sequences that could arise from V-J or V-D-J coupling. For CDR3 length 3, 4, or 5, we start with the amino-acid sequences shown in Table 7. For example, Sequence V-3JH1 shows the C-terminal end of 3-23 FR3 (TAVYYCAK (SEQ ID NO:30)) followed by JH1 which has been trimmed from the N-terminal end until three amino-acids before the Trp-Gly that starts FR4. V-3JH2 shows the end of FR3 followed by the trimmed JH2. The sequence following V-3JH6 are constructed by joining FR4 to a trimer taken from a human D segment followed by the FR4 region of a human JH segment. 3D3-3.3.2 would be a trimer from segment D3-3, third reading frame starting at the second amino acid. 5D5-12.2.3 is a pentamer from D5-12 in reading frame 2 starting at amino acid 3. Some of the germ-line D segments contain stop codons, yet they appear in natural antibodies when the stop codons are edited away. Here we assume that the most likely change fro TAA and TAG codons is to Tyr (Y) and that TGA stops are most likely mutated to Trp (W). Table 20 shows the amino-acid sequences of the human D segments; the types of stop codons is indicated by the use of * for TAG, @ for TAA, and $ for TGA. In Table 11 are 266 distinct trimers that can be constructed from human D segments. The TAA and TAG stops have been changed to Tyr shown as “y” (i.e., lowercase). These could also be changed to Ser, Cys, Phe, Gln, Lys, or Glu by single base changes. TAG could be changed by single base changes to Trp as well as Tyr, Gln, Lys, Glu, Ser, and Leu. Table 12 shows the 266 distinct tetramers that can be obtained by trimming human D segments. Table 13 shows the 215 pentamers that can be obtained from trimming human D segments. Table 14 shows the 155 hexamers that can be obtained by trimming human D segments. The libraries to be built have substantial diversity in HC CDR1 and HC CDR2. The sequence diversity of HC CDR3 may be less important than having a short, but acceptable sequence. The diversity of JH segments or fragments (e.g., 3 or more amino acids) of D segments provides sequences that could be built by the human immune system and so are less likely to be immunogenic.

In one embodiment, the trimers, tetramers, and pentamers that contain a Cys are eliminated.

In one embodiment, the trimers, tetramers, and pentamers that contain a Cys or the came from a D fragment containing a stop are eliminated.

The short libraries constructed using the trimers of Table 11, tetramers of Table 12, pentamers of Table 13 have substantial diversity: 266, 266, and 215 respectively. This is to be compared to the number of peptides of these lengths: 8000, 160000, and 3200000 respectively.

V-3D1-1.1.1-JH1 contains the final portion of FR3 followed by three amino acids from D1-1 (RF1), viz. GTT (SEQ ID NO:257). V-3D1-1.2-JH1 uses amino acids 2-4 of D1-1 (RF1) as the parental CDR3. V-3D3-3.3.3-JH2 shows the end of FR3 followed by amino acids 3-5 of D3-3 (RF 3). The invention comprises any amino-acid sequence comprising FR3::(three, four, or five stop-free AAs of a human D segment)::FR4 from a human JH. Fragments of D regions containing unpaired Cys residues are less preferred than those that are free of unpaired Cys residues. In V-5JH3, there is a Tyr shown as ‘y’ because JH3 has only 4 codons before the codons for Trp-Gly that define the beginning of FR4. V-5JH4 has a Ser shown as ‘s’ for the same reason. If wobbling is used, the preferred level of purity is between 0.75 and 0.90. The invention comprises the sequences V-3JH1 through V-3JH6, V-4JH1 through V-4JH6, and V-5JH1 through V-5JH6, and libraries containing the same The invention also comprises the sequences in which the CDR region is replaced by a 3, 4, or 5 amino-acid segment from a human D region, and libraries containing the same. The invention further comprises DNA in which the parental sequence has been mutated in the CDR3 region, and libraries containing the same. A preferred embodiment is one in which the average number of base changes per CDR3 is one, two, or three. The methods of mutagenesis include error-prone PCR, wobbling, and dobbling.

TABLE 7 Amino-acid sequences of parental CDR3s of lengths 3, 4, 5 ...FR3----- CDR3- FR4-------- Length 3 V-3JH1    TAVYYCAK   FQH WGQGTLVTVSS (SEQ ID NO: 31) V-3JH2    TAVYYCAK   FDL WGRGTLVTVSS (SEQ ID NO: 32) V-3JH3    TAVYYCAK   FDI WGQGTMVTVSS (SEQ ID NO: 33) V-3JH4    TAVYYCAK   FDY WGQGTLVTVSS (SEQ ID NO: 34) V-3JH5    TAVYYCAK   FDP WGQGTLVTVSS (SEQ ID NO: 35) V-3JH6    TAVYYCAK   MDV WGQGTTVTVSS (SEQ ID NO: 36) V-3D1-1.1.1-JH1    TAVYYCAK   GTT WGQGTLVTVSS (SEQ ID NO: 37) V-3D1-1.1.2-JH1    TAVYYCAK   TTG WGQGTLVTVSS (SEQ ID NO: 38) V-3D3-3.3.3-JH2    TAVYYCAK   IFG WGRGTLVTVSS (SEQ ID NO: 39) Length 4 V-4JH1    TAVYYCAK  YFQH WGQGTLVTVSS (SEQ ID NO: 40) V-4JH2    TAVYYCAK  YFDL WGRGTLVTVSS (SEQ ID NO: 41) V-4JH3    TAVYYCAK  AFDI WGQGTMVTVSS (SEQ ID NO: 42) V-4JH4    TAVYYCAK  YFDY WGQGTLVTVSS (SEQ ID NO: 43) V-4JH5    TAVYYCAK  WFDP WGQGTLVTVSS (SEQ ID NO: 44) V-4JH6    TAVYYCAK  GMDV WGQGTTVTVSS (SEQ ID NO: 45) V-4D3-10.1a-JH2    TAVYYCAK  LLWF WGRGTLVTVSS (SEQ ID NO: 46) Length 5 V-5JH1    TAVYYCAK EYFQH WGQGTLVTVSS (SEQ ID NO: 47) V-5JH2    TAVYYCAK WYFDL WGRGTLVTVSS (SEQ ID NO: 48) V-5JH3    TAVYYCAK yAFDI WGQGTMVTVSS (SEQ ID NO: 49) V-5JH4    TAVYYCAK sYFDY WGQGTLVTVSS (SEQ ID NO: 50) V-5JH5    TAVYYCAK NWFDP WGQGTLVTVSS (SEQ ID NO: 51) V-5JH6    TAVYYCAK YGMDV WGQGTTVTVSS (SEQ ID NO: 52) V-5D2-8.2a-JH2    TAVYYCAK DIVLM WGRGTLVTVSS (SEQ ID NO: 53)

TABLE 8 DNA encoding V-5D2-8.2a-JH2 for wobbling !                                               CDR3....... !   A   E   D   T   A   V   Y   Y   C   A   K   D   I   V   L   M |gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aag jez ezq jzz qzz ezj ! !  W   G   Q   G   T   T   V   T   V   S   S (SEQ ID NO: 54) tgg ggc cag ggt act acG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 55) !                 BstEII...

Alternatively, one could synthesize three fragments of DNA that correspond to the region from XbaI to BstEII and having residue 94 being K or R followed by 3, 4, or 5 NNK codons, followed by WG . . . of FR4. The allowed variation is 20³+20⁴+20⁵=3,368,000. After amplification, these DNA molecules would be mixed in the ratio 1:10:100 (so that shorter sequences are relatively oversampled) and cloned into the phagemid encoding the kappa library with HC CDR1/2 diversity. A library of 1×10⁹ would give significant diversity and will allow isolation of antibodies that bind to targets that have small to medium protrusions. For example, various carbohydrates, loops of proteins that are not well ordered (such as GPCRs) may benefit from a groove in the antibody created by having a very short HC CDR3. We can also build a lambda library. The ratio of AA sequences is 1:20:400, and it may be important to sample the shorter sequences more densely. Getting a big, wide gulley in the Ab may require exactly one 3 AA CDR3, but with a 4 AA CDR3, one probably has more leeway and with 5 AAs, even more leeway. In this Example, we use the JH6 version of FR4 from the WG motif onward.

We can select from our current kappa library a collection of, for example, 25 kappa light chains that are a) germline in the framework regions, b) show suitable diversity in CDRs, and c) are of types that produce well and pair well with 3-23. These LC_(s) will be made in E. coli from a vector that carries Kan^(R) and no phage packaging signal. We would then build our HC library in a phage vector that has no LC. HC and LC will be crossed by infecting the LC producing cells with the HC phage. HC phage that are selected can be combined with the LC of the cell that produces ELISA phage or the HCs can be cloned into pMID21 that have the whole LC diversity. Alternatively, the selected HC can be moved into pHCSK85 and used with ROLIC to combine with all the LCs of our collection. Lambda LCs could also be used. Thus, a library of 1×10⁹ HC in phage can be expanded into a Fab library of 1.2×10¹¹ (1×10⁹×117). If we combined 1×10⁷ CDR1-2s with 10⁶ HC CDR3s, we could make a library of 5×10⁷ in which each CDR3 is coupled with 50 CDR1-2s. A library of 5×10⁷ HCs in phage could give results similar to an old-style library of 6×10⁹.

TABLE 1 Designs of very short exemplary HC CDR3s C3XXX !  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S 5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC- !              XbaI... ! !                                                      CDR3....... !   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R any any any  W   G   |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg nnk nnk nnk tgg ggc- ! !   Q   G   T   T   V   T   V   S   S (SEQ ID NO: 56)    cag ggt act acG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 57) !                BstEII... ! (C3XXX)5′-T|GCA|GtT|taT|taC|tgc|gct aRg nnk nnk nnk tgg ggc cag ggt act ac-3′ (SEQ ID NO: 58) (ON_5) 5′-AcTggAgAcggTgAccgTAgTAcccTggccccA-3′ ! 33 bases (SEQ ID NO: 256) (ON_5 is reverse complement of 5′-tgg ggc cag ggt act acG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 59)) ! Use ON-1 and ON-3 shown below !----------------------------------------------- ! C3X4 !  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S 5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC- !              XbaI... ! !                                                      CDR3........... !   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R any any any any  W   |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg nnk nnk nnk nnk tgg- ! !   G   Q   G   T   T   V   T   V   S   S (SEQ ID NO: 60)    ggc cag ggt act acG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 61) !                    BstEII... ! (C3X4)5′-GCA|GtT|taT|taC|tgc|gct aRg nnk nnk nnk nnk tgg-            ggc cag ggt act ac-3′ (SEQ ID NO: 62) ! Use ON-1, ON-3, and ON-5 !---------------------------------------------------------- C3X5 !  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S 5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC- !              XbaI... ! !                                                      CDR3............... !   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R any any any any any   |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg nnk nnk nnk nnk nnk- ! !   W   G   Q   G   T   T   V   T   V   S   S (SEQ ID NO: 63)    tgg ggc cag ggt act acG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 64) !                        BstEII... (C3X5)5′-GCA|GtT|taT|taC|tgc|gct aRg nnk nnk nnk nnk nnk tgg-            ggc cag ggt act ac-3′ (SEQ ID NO: 65) !------------------------------------------------- aRg encodes K or R

Alternatively, the current HC diversity can be cloned into DY3F87HC and the CDR3 diversity described above is cloned into that diversity as XbaI-BstEII fragments. A library of, for example, 25 LC are cloned into pLCSK23 and used to create a cell line in TG1 E. coli. These cells are infected with the DY3F87HC phage which harbor the novel HC CDR3 (and CDR1-2) diversity. The phage obtained from this infection are selected for binding to a desired target. After two to four rounds of selection, the selected HCs are transfered to pHCSK22 and used to create a cell line which can be used with ROLIC to combine the selected HC with all the LCs in the ROLIC LC library. In this way, a library of 1. E 9 can be give Abs that normally would require construction of a library of 1. E 16 (assuming a LC diversity of 1. E 7).

Prophetic Example 2: Libraries with Very Long HC CDR3s

Sidhu et al. (J Mol Biol. 2004 338:299-310. and US application 20050119455A1) report high-affinity Abs selected from a library in which only Y and S were allowed in the CDRs which were limited in length to 20 amino acids. It may be possible to generate high affinity Abs from a library that has HC CDR3s with one or more of the following forms of diversity: a) several (but not all) sites allowing Y or S, b) including 4-6 NNK codons, c) introducing D segments (with or without diversification in the D), and/or d) using error-prone PCR. We have already sampled the Ab space in which HC CDR3 is in the range ˜8 to ˜22 with a median length of 13. Thus, libraries in which HC CDR3 is either ˜23 AAs or ˜35 AAs are possible and may have advantages with certain types of targets. For example, GPCRs are integral membrane proteins with seven helical segments transversing the lipid bilayer of the call that are thought to have multiple states. An antibody having a very long HC CDR3 could form a protuberance that fits into the channel formed by the seven strands. Finding Abs that bind GPCRs has been difficult and intentionally building libraries in which all the members have very long HC CDR3s may ameliorate this problem. The lengths may be made somewhat variable, say 23, 24, or 25 in one library and 33, 34, or 35 in a second.

Below are a number of representative designs. The CDR3 have been broken up and diversity generated that lets the various parts have differing relationships depending on the value of X. A full-length JH1 has been used, and in some designs diversity allowed diversity in the CDR3 part of JH1. Other JHs could be used. In the designs, the D segments are either rich in Y or have an S-rich disulfide loop. The amino-acid sequences of human D segments are shown in Table 3. The places where the D region has either S or Y or allowed other combinations have in particular been varied. Table 4 shows the amino-acid sequences of human J regions.

Each of the libraries could be built in at least four ways: 1) DNA encoding a particular amino acid sequence is first synthesized and subjected to error-prone PCR, 2) the library can be synthesized by wobbling or with mixtures of nucleotides, 3) the library can be built using dobbling, and 4) routes (2) or (3) could be followed by error-prone PCR. As an example of route (1), in Design 12, DNA encoding SEQ ID NO:908 could be synthesized, as shown in SEQ ID NO:911. This DNA could be subjected to error-prone PCR using the primers shown in SEQ ID NO:909 and SEQ ID NO:910. Because these primers cover the framework regions, the errors will occur only in the CDR3.

A library of HCs with CDR3 with length 23 of, for example, 2×10⁹ members and a second library with HC CDR3s of length ˜35 also having 2×10⁹ members could be built. Alternatively, the DNA could be mixed to build one library of 4×10⁹.

TABLE 4 Human JH amino-acid sequences   H3 ------   CDR3 --------     100       110       |         | JH1 ---AEYFQHWGQGTLVTVSS (SEQ ID NO: 66) JH2 ---YWYFDLWGRGTLVTVSS (SEQ ID NO: 67) JH3 -----AFDIWGQGTMVTVSS (SEQ ID NO: 2) JH4 -----YFDYWGQGTLVTVSS (SEQ ID NO: 1) JH5 ----NWFDPWGQGTLVTVSS (SEQ ID NO: 68) JH6 YYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 3)

In each of the following designs, the amino-acid sequence begins with YYCA(K/R) (SEQ ID NO: 936) which is the end of FR3. FR4 starts with WG and is shown bold.

Design 1

SEQ ID NO:898 comprises the end of FR3 joined to two residues (DY) of types often found in the filler sequence that the immune system places between V and D. These are followed by D2-2.2, preferred because it has a disulfide loop and is rich in Ser and Tyr residues. This is followed by YGYSY (SEQ ID NO: 937), which is rich in Tyr and Ser residues, which is followed by full-length JH1.

XX::D2-2.2::XX::JH1                1    1    2  2   FR3 1   5    0    5    0  3FR4 YYCAK DYGYCSSTSCYTYGYSYAEYFQHWGQGTLVTVSS (SEQ ID NO: 898) YYCAK XX GYCSXXSCYT XXYSYAEYFQHWGQGTLVTVSS (SEQ ID NO: 69)     R   GYCSSTSCYT     AEYFQHWGQGTLVTVSS (JH1)        (SEQ ID NO: 70)    (SEQ ID NO: 66)            1 1               1     1     9 9    0 0               0     1     4 5    0 2abcdefghijklmnp3     0 Amino-acid diversity = 1.28 E 8 DNA diversity = 2.15 E 9 Stop-free = 83% Gratuitous Cys-free = 83% Free of stop and Cys = 68%

Design 1(C23D222) has 94 being R or K, then 2 Xs, D2-2 in second reading frame with two Xs in the loop, followed by two Xs, and JH1. D2-2 2^(nd) reading frame has a disulfide-closed loop into which diversity at two points has been introduced. This CDR3 is 23 long. Using primers that include DNA up to . . . YYCA (SEQ ID NO: 938) and from WGQG (SEQ ID NO: 939) . . . , error-prone PCR on the CDR3 could be performed before amplifying out to XbaI and BstEII for cloning into the library of kappa LC and HC CDR1/2. Thus, the AAs that are shown as fixed will be allowed to vary some. The AAs that are part of the PCR overlap region will be reinforced by the final non-error prone PCR. Error-prone PCR is not a necessary part of the design.

C23D222JH1 !  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S 5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC- !              XbaI... ! !   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R   |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - ! ! CDR3---------------------------------------------------------------- !  X   X   D2-2 RF2.............................   X   X              JH1.. !  any any  G   Y   C   S  any any  S   C   Y   T  any any  Y   S   Y   A    nnk nnk ggt tat tgt tcc nnk nnk tct tgc tat act nnk nnk tat tcc tac gct- ! !  CDR3--------------- !   E   Y   F   Q   H    gaa tat ttc cag cac- ! !   W   G   Q   G   T   L   V   T   V   S   S  (SEQ ID NO: 71)    tgg ggc cag ggt act ctG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 72) !                        BstEII... (ON_C23D222)  5′-GCA|GtT|taT|taC|tgc|gct aRg nnk nnk ggt tat tgt tcc nnk-       nnk tct tgc tat act nnk nnk tat tcc tac gct gaa tat ttc cag cac-       tgg ggc cag ggt act ct-3′ ! 107 bases (SEQ ID NO: 73) (ON_1) 5′-GCA|GtT|taT|taC|tgc|gct-3′ (SEQ ID NO: 74) (ON_2) 5′-AgAgTAcccTggccccAgAcgTccATAccgTAATAgT-3′ ! 37 bases (SEQ ID NO: 75) (ON_2 is reverse complement of 5′-ac tat tac ggt atg gac gtc tgg ggc cag ggt act ct-3′) (SEQ ID NO: 76) (ON_3) 5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg-           aac|agC|TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct-3′ (SEQ ID NO: 77) (ON_4) 5′-AcTggAgAcggTgAccAgAgTAcccTggccccA-3′ ! 33 bases (SEQ ID NO: 78) (5′-tgg ggc cag ggt act ctG GTC ACC gtc tcc agt-3′ [RC] (SEQ ID NO: 79)) Design 2

               1    1    2  2       1   5    0    5    0  3 YYCAK GSYYYGSGSYYNVDSYYAEYFQHWGQGTLVTVSS (SEQ ID NO: 899) YYCAK XXYYYGXGSXYNXXSYYAEYFQHWGQGTLVTVSS  (SEQ ID NO: 80)     R   YYYGSGSYYN     AEYFQHWGQGTLVTVSS (JH1)        (SEQ ID NO: 81) (SEQ ID NO: 66) Amino-acid diversity = 1.28 E 8 DNA diversity = 2.15 E 9 Stop-free = 83% Gratuitous Cys-free = 83% Free of stop and Cys = 68%

Design 2 (C23D310) has 94 as R or K, two Xs, D3-10 (RF2) with 5^(th) and 8^(th) residues changed to X, 2 Xs, SYY, and JH1. The CDR3 is 23 AA long and could be further diversified by use of error-prone PCR.

C23D310JH1 !  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S 5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC- !              XbaI... ! !   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R   |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - ! ! CDR3------------------------------------------------------------------- ! !  any any  Y   Y   Y   G  any  G   S  any  Y   N  any any  S   Y   Y    nnk nnk tac tac tat ggt nnk ggc tct nnk tac aat nnk nnk tct tat tac ! !   A   E   Y   F   Q   H    gct gag tac ttt caa cat ! !   JH1...................................... !   W   G   Q   G   T   L   V   T   V   S   S (SEQ ID NO: 82)    tgg ggc cag ggt act ctG GTC ACC gtc tcc agt-3′ (SEQ ID NO: 83) !                        BstEII... (C23D310) 5′-GCA|GtT|taT|taC|tgc|gct aRg nnk nnk tac tac tat ggt nnk ggc-  tct nnk tac aat nnk nnk tct tat tac gct gag tac ttt caa cat tgg ggc cag-  ggt act ct-3′ (SEQ ID NO: 84) ON_1, ON_2, ON_3, and ON_4 as above. Design 3

               1    1    2  2       1   5    0    5    0  3 YYCAK DYYYYGSGSYYNSDSYSAEYFQHWGQGTLVTVSS (SEQ ID NO: 900) YYCAK XZ YZZGZGZXYN ZXZYZ AXZFQHWGQGTLVTVSS (SEQ ID NO: 940)     R   YYYGSGSYYN     AEYFQHWGQGTLVTVSS (JH1)        (SEQ ID NO: 81) (SEQ ID NO: 66) Amino-acid diversity = 1.64 E 8 DNA diversity = 1.07 E 9 Stop-free = 88% Gratuitous Cys-free = 88% Free of stop and Cys = 77%

Design 3 (C23D310B) has 94 as R or K, XZ, D3-10 (RF2) with 2^(nd), 3^(rd), 5^(th), and 7^(th) as Z(YIS) and 8^(th) residue changed to X, ZXZYZ(SEQ ID NO:992), and JH1 (with the E changed to X). Z is either Y or S. The CDR3 is 23 AA long and could be further diversified by use of error-prone PCR.

               A   V   Y   Y   C   A  R|K any Y|S  Y  Y|S Y|S  G  Y|S  G (C23D310b) 5′-GCA|GtT|taT|taC|tgc|gct aRg nnk tmc tac tmc tmt ggt tmc ggc-   Y|S any  Y   N  Y|S any Y|S  Y  Y|S  A  any Y|S  F   Q   H   W   G   Q   tmt nnk tac aat tmt nnk tmc tat tmc gct nnk tmc ttt caa cat tgg ggc cag-    G   T   L     (SEQ ID NO: 85)   ggt act ct-3′     (SEQ ID NO: 86)

ON_1, ON_2, ON_3, and ON_4 as above.

Design 4

               1    1    2  2 2    3    3       1   5    0    5    0  3 5    0    5 YYCAK YYSFSYYPYYYDSSGYYYGYYSDYSYSYYAEYFQHWGQGTLVTVSS  (SEQ ID NO: 901) YYCAK YYSXSYYX YZYDSZGYZY XYYSXYZYZZZ AZZFQHWGQGTLVTVSS   (SEQ ID NO: 87)     R         YYYDSSGYYY           AEYFQHWGQGTLVTVSS (JH1)               (SEQ ID NO: 88)         (SEQ ID NO: 66)            1 1                           1     1     9 9    0 0                           0     1     4 5    0 2abcdefghijklmnopqrstuvwxyab3     0                                        ′′ Amino-acid diversity = 1.64 E 8 DNA diversity = 1.07 E 9 Stop-free = 88% Gratuitous Cys-free = 88% Free of stop and Cys = 77%

Design 4 has CDR3 of length 35. Residue 94 can be K or R, then YYS::X::SYY::X::D3-22(2^(nd) RF with one S as X and 3 Zs)::X::YYS::X::YZZZ(SEQ ID NO:993)::JH1(with 2 Zs). Error-prone PCR could be used to add more diversity.

C35D322JH1 ! scab  DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S 5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC- !              XbaI... ! !   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R   |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - ! !  CDR3------------------------------------------------------------------- ! !   Y   Y   S  any  S   Y   Y  any  Y  Y|S  Y   D   S  Y|S  G   Y  Y|S  Y    tac tat tcc nnk tct tac tat nnk tat tmt tac gat agt tmt ggt tac tmc tat !    any  Y   Y   S  any  Y  Y|S  Y  Y|S Y|S Y|S  A  Y|S Y|S  F   Q   H    nnk tac tat agc nnk tat tmc tac tmc tmt tmc gct tmt tmc ttc caa cac ! !   W   G   Q   G   T   L   V   T   V   S   S     (SEQ ID NO: 89)    tgg ggc cag ggt act ct G GTC ACC gtc tcc agt-3′   (SEQ ID NO: 90) !                        BstEII... (c35d322B) 5′-GCA|GtT|taT|taC|tgc|gct aRg tac tat tcc nnk tct tac tat nnk-   tat tmt tac gat agt tmt ggt tac tmc tat nnk tac tat agc nnk tat tmc tac-   tmc tmt tmc gct tmt tmc ttc caa cac tgg ggc cag ggt act ct-3′   (SEQ ID NO: 91) ON_1, ON_2, ON_3, and ON_4 as above. Design 5

               1    1    2  2       1    5   0    5    0  3 YYCAK SSGYCSSTSCYTGVYYYAEYFQHWGQGTLVTVSS (SEQ ID NO: 902) YYCAK ZZ GZCZZXZCZT XXYZYX ZYFQHWGQGTLVTVSS     (SEQ ID NO: 92)     R   GYCSSTSCYT     AEYFQHWGQGTLVTVSS (JH1)        (SEQ ID NO: 70)  (SEQ ID NO: 66) Amino-acid diversity = 1.64 E 8 DNA diversity = 1.07 E 9 Stop-free = 88% Gratuitous Cys-free = 88% Free of stop and Cys = 77%

Design 5(C23D222b) is like design 1 but uses many Z(Y or S) variable codons. This CDR3 is 23 long.

C23D222JH1b !  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S 5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC- !              XbaI... ! !   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R   |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - ! !  CDR3------------------------------------------------------------------- !  Y|S Y|S  G  Y|S  C  Y|S Y|S any Y|S  C  Y|S  T  any any  Y  Y|S  Y  any    tmc tmt ggt tmt tgc tmc tmt nnk tmt tgt tmc acc nnk nnk tat tmt tac nnk ! !  Y|S  Y   F   Q   H    tmt tat ttc cag cac ! !   W   G   Q   G   T   L   V   T   V   S   S    (SEQ ID NO: 93)    tgg ggc cag ggt act ctG GTC ACC gtc tcc agt-3′    (SEQ ID NO: 94) !                        BstEII... (C23D222JH1b) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmc tmt ggt tmt tgc tmc tmt-  nnk tmt tgt tmc acc nnk nnk tat tmt tac nnk tmt tat ttc cag cac tgg ggc-  cag ggt act ct-3′     (SEQ ID NO: 95) Design 6

               1    1    2  2 2    3    3       1   5    0    5    0  3 5    0    5 YYCAK SYDYYGYCSSTSCYTYYSYVSYSSYYSYYAEYFQHWGQGTLVTVSS  (SEQ ID NO: 903) YYCAK ZYXZYGZCZZXSCZTYZSZXZYSZYZSZYAEZFQHWGQGTLVTVSS      (SEQ ID NO: 96)     R      GYCSSTSCYT D2-2.2       AEYFQHWGQGTLVTVSS (JH1)             (SEQ ID NO: 70)          (SEQ ID NO: 66) Amino-acid diversity = 2.00 E 8 DNA diversity = 5.37 E 8 Stop-free = 91% Gratuitous Cys-free = 91% Free of stop and Cys = 83% C35D222JH1 ! !  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S 5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC- !              XbaI... ! !   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R   |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - ! !  CDR3------------------------------------------------------------------- !  Y|S  Y  any Y|S  Y   G  Y|S  C  Y|S Y|S any  S   C  Y|S  T   Y  Y|S  S    tmt tac nnk tmc tac ggc tMt tgc tmt tmc nnk tCt tgt tmc acc tat tmt tcc ! !  Y|S any Y|S  Y   S  any  Y  Y|S  S  Y|S  Y   A   E   Y   F   Q   H    tmt nnk tmc tat tct nnk tac tmc agt tmt tat gct gag tat ttc cag cac ! !   W   G   Q   G   T   L   V   T   V   S   S    (SEQ ID NO: 97)    tgg ggc cag ggt act ctG GTC ACC gtc tcc agt-3′     (SEQ ID NO: 98) !                        BstEII... (C35D222JH1)5′-GCA|GtT|taT|taC|tgc|gct aRg tmt tac nnk tmc tac ggc tat- tgc tmt tmc nnk tmt tgt tmc acc tat tmt tcc tmt nnk tmc tat tct nnk tac- tmc agt tmt tat gct gag tat ttc cag cac tgg ggc cag ggt act ct-3′  (SEQ ID NO: 99) Design 7

               1    1    2  2 2    3    3       1   5    0    5    0  3 5    0    5 YYCAK YYSYYGYCSSTSCYTYSSSVSYSYYSSYYAEYFQHWGQGTLVTVSS   (SEQ ID NO: 904) YYCAK ZYZZYGZCZZXZCZTYZSZXZYSZYZSZYAψZJQBWGQGTLVTVSS  (SEQ ID NO: 100)     R      GYCSSTSCYT D2-2.2       AEYFQHWGQGTLVTVSS (JH1)             (SEQ ID NO: 70)          (SEQ ID NO: 66) (J = FSY, B = YHND, ψ = EKQ)   Amino-acid diversity = 9.44 E 8 DNA diversity = 2.42 E 9 Stop-free = 93% Gratuitous Cys-free = 93% Free of stop and Cys = 88% C35D222JH1B !  !  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S 5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC- !              XbaI... ! !   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R   |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - ! !  CDR3---------------------------------------------------------------- !  Y|S  Y  Y|S Y|S  Y   G  Y|S  C  Y|S Y|S any Y|S  C  Y|S  T   Y  Y|S  S    tmt tac tmc tmc tac ggc tMt tgc tmt tmc nnk tmt tgt tmc acc tat tmt tcc ! !                                                   Q       Y      N|D !  Y|S any Y|S  Y   S  Y|S  Y  Y|S  S  Y|S  Y   A  E|K Y|S F|S  Q  H|Y    tmt nnk tmc tat tct tmt tac tmc agt tmt tat gct Vag tmt tHc cag Nac ! !   W   G   Q   G   T   L   V   T   V   S   S  (SEQ ID NO: 101)    tgg ggc cag ggt act ctG GTC ACC gtc tcc agt-3′   (SEQ ID NO: 102) !                        BstEII... Design 8

               1    1    2  2 2    3    3       1   5    0    5    0  3 5    0    5 YYCAK SRSYYDYVWGSYRYTSSYSYYSYSYSSYAEYFQHWGQGTLVTVSS  (SEQ ID NO: 905) YYCAK ZXZ YZBZVWGZZRZT ZSZXZYZZZYZSZ AψZFQHWGQGTLVTVSS  (SEQ ID NO: 103)     R    YYDYVWGSYRYT D3-16.2     AEYFQHWGQGTLVTVSS (JH1)             (SEQ ID NO: 104)          (SEQ ID NO: 66) (J = FSY, B = YHND, ψ = EKQ) Amino-acid diversity = 9.44 E 8 DNA diversity = 1.61 E 9 Stop-free = 93% Gratuitous Cys-free = 93% Free of stop and Cys = 88% C34D316JH1A ! !  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S 5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC- !              XbaI... ! !   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R   |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - ! !  CDR3---------------------------------------------------------------- !                      N|D !  Y|S any Y|S  Y  Y|S Y|H Y|S  V   W   G  Y|S Y|S  R  Y|S  T  Y|S    tmt nnk tmc tac tmt Nat tmt gtt tgg ggt tmt tmc cgt tmt act tmt ! !   S  Y|S any Y|S  Y  Y|S Y|S Y|S  Y  Y|S  S  Y|S    agt tmc nnk tmt tac tmc tmt tmc tat tmc agt tmt ! !        Q !    A  E|K Y|S  F   Q   H     GCT vag tmc ttc cag cat ! !   W   G   Q   G   T   L   V   T   V   S   S   (SEQ ID NO: 105)    tgg ggc cag ggt act ct G GTC ACC gtc tcc agt-3′    (SEQ ID NO: 106) !                        BstEII... (C34D316JH1A) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmt nnk tmc tac tmt Nat tmt-  gtt tgg ggt tmt tmc cgt tmt act tmt agt tmc nnk tmt tac tmc tmt tmc tat-  tmc agt tmt GCT vag tmc ttc cag cat tgg ggc cag ggt act ct -3′  (SEQ ID NO: 107) Design 9

Design 9 is like 8 except the D segment is moved to the right

               1    1    2  2 2    3    3       1   5    0    5    0  3 5    0    5 YYCAK YGYSSDSYYSSYYDYVWGSYRYTYSSYYAEYFQHWGQGTLVTVSS (SEQ ID NO: 906) YYCAK ZXZZZXZYZZZYZBZVWGZZRZTYZSZYAψZFQHWGQGTLVTVSS  (SEQ ID NO: 108)     R  D3-16.2   YYDYVWGSYRYT     AEYFQHWGQGTLVTVSS (JH1)                 (SEQ ID NO: 104)  (SEQ ID NO: 66) (J = FSY, B = YHND, ψ = EKQ) Amino-acid diversity = 1.31 E 8 DNA diversity = 5.37 E 8 Stop-free = 91% Gratuitous Cys-free = 91% Free of stop and Cys = 83% C34D316JH1B ! !  scab DNA     S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S 5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC- !              XbaI... ! !   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R   |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - ! !  CDR3-------------------------------------------------------------------    Y|S any Y|S Y|S Y|S any Y|S  Y  Y|S Y|S Y|S    tmt nnk tmc tmt tmc nnk tmt tac tmc tmt tmc ! !          N|D !   Y  Y|S Y|H Y|S  V   W   G  Y|S Y|S  R  Y|S  T    tac tmt Nat tmt gtt tgg ggt tmt tmc cgt tmt act ! !   Y  Y|S  S  Y|S  Y    tat tmc agt tmt tac !  !        Q !    A  E|K Y|S  F   Q   H     GCT vag tmc ttc cag cat ! !   W   G   Q   G   T   L   V   T   V   S   S    (SEQ ID NO: 109)    tgg ggc cag ggt act ct G GTC ACC gtc tcc agt-3′    (SEQ ID NO: 110) !                        BstEII... (C35D316JH1B) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmt nnk tmc tmt tmc nnk tmt tac tmc tmt tmc tac tmt Nat tmt gtt tgg ggt tmt tmc cgt tmt act tat tmc agt tmt tac GCT vag tmc ttc cag cat tgg ggc cag ggt act ct-3′    (SEQ ID NO: 111) Design 10

               1    1    2   2       1   5    0    5    0   4 YYCAK GSSYYYGSGSYYNSDYYSAEYFQHWGQGTLVTVSS  (SEQ ID NO: 907) YYCAK XZZ YZZGZGZXYN ZXZYZ AXZFQHWGQGTLVTVSS     (SEQ ID NO: 112)     R    YYYGSGSYYN     AEYFQHWGQGTLVTVSS (JH1)         (SEQ ID NO: 81)   (SEQ ID NO: 66)

Design 10 (C24D310B) is like Design 3, but the CDR3 is of length 24. Design 10 has 94 as R or K, XZZ, D3-10 (RF2) with 2^(nd), 3^(rd), 5^(th), and 7^(th) as Z(YIS) and 8^(th) residue changed to X, ZXZYZ(SEQ ID NO: 994), and JH1 (with the E changed to X). Z is either Y or S. The CDR3 is 24 AA long and could be further diversified by use of error-prone PCR.

(C24D310b) 5′-GCA|GtT|taT|taC|tgc|gct aRg nnk tmc tmc tac tmc tmt ggt tmc-   ggc tmt nnk tac aat tmt nnk tmc tat tmc gct nnk tmc ttt caa cat tgg ggc-   cag ggt act ct-3′    (SEQ ID NO: 113)

ON_1, ON_2, ON_3, and ON_4 as above.

Design 11

               1    1    2   2       1   5    0    5    0   5 YYCAR SSRSGYCTNGVCYRSGSYWYFDLWGRGTLVTVSS  (SEQ ID NO: 991) YYCAR ZZXZGZC32GVCZ3ZXZZ4Z12LWGRGTLVTVSS    (SEQ ID NO: 114)     K     GYCTNGVCYT   YWYFDLWGRGTLVTVSS  D2-8.2  JH2          (SEQ ID NO: 115)   (SEQ ID NO: 67) (1 = FYS(THT), 2 = YHND(NAT), 3 = ITKR(ANA), 4 = LSW(TBG)) (C24D282) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmc tmt nnk tmt ggt tmc tgt ana-  nat ggt gtc tgc tmt ana tmc nnk tmt tmt tbg tmt tht nat ctg tgg ggc-   cag ggt act ct-3′    (SEQ ID NO: 116) (C24D282.1) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmc tmt nnk tmc ggt tmc tgc ana-  nat ggc gtc tgc tmt ana tmc nnk tmt tmt tbg tmt tht nat ctg tgg ggc-   cag ggt act ct-3′    (SEQ ID NO: 117) (C24D282.1) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmc tmt nnk tm c ggt tmc tgc ana-  nat ggc gtc tgc t -3′    (SEQ ID NO: 118) (needs R, M, N, K) (C24D282.2) 5′-Ag AgT Acc cTg gcc ccA cAg ATN ADA AKA cVA AKA AKA MNN gKA TNT AKA gcA gAc gcc ATN TNT gcA gKA Acc g-3′    (SEQ ID NO: 119) ! 75 bases (5′- c ggt tmc tgc ana-  nat ggc gtc tgc t mt ana tmc nnk tmt tmt tbg tmt tht n at ctg tgg ggc-   cag ggt act ct-3′ [RC]    (SEQ ID NO: 120) (needs N, M, K, B, H)) Design 12

               1    1    2    2    3    3       1   5    0    5    0    5    0    5 YYCAR SSYYSYGYCTNGVCYTYSYSYYSYSYSYWYFDLWGRGTLVTVSS (SEQ ID NO: 908) YYCAR ZZZZZZGZC32GVCZ3ZZZZYZZYZYZZ4Z12LWGRGTLVTVSS    (SEQ ID NO: 121)     K       GYCTNGVCYT           YWYFDLWGRGTLVTVSS  D2-8.2  JH2            (SEQ ID NO: 115)      (SEQ ID NO: 67) (1 = FYS, 2 = YHND, 3 = ITKR, 4 = LSW, Z = YS) (C33D282TP) 5′-GCA|GtT|taT|taC|tgc|gct-3′ (SEQ ID NO: 909) (C33D282BP) 5′-ag agt acc ctg gcc cca-3′ (SEQ ID NO: 910) (C33D282) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmt tmc tmc tmt tmc tmc ggt-   tmt tgt ana nat ggc gtg tgc tmt ana tmc tmc tmc tmt tat tmt tmc tat tmt-   tac tmt tmc tbg tmc tht nat ctg tgg ggc cag ggt act ct-3′(SEQ ID NO: 122) (C33D282F) 5′-GCA|GtT|taT|taC|tgc|gct agg tct tcc tac tat tcc tac ggt-   tat tgt aca aat ggc gtg tgc tat aca tac tcc tac tct tat tat tcc tat tct-   tac tct tac tgg tac ttt gat ctg tgg ggc cag ggt act ct-3′(SEQ ID NO: 911) Design 13

Design 13 places a germ-line D segment in the middle of a sea of Zs so that one can make two pieces of DNA that overlap throughout the constant region. HC CDR3 is 34 long and diversity is 2²³ ˜8×10⁶.

               1    1    2    2    3    3       1   5    0    5    0    5    0    5 YYCAR SSSYYSYYSSGYCTNGVCYTYSSYYSSYYWYFDLWGRGTLVTVSS (SEQ ID NO: 912) YYCAR ZZZZZZZZZZGYCTNGVCYTZZZZZZZZZWZF2LWGRGTLVTVSS    (SEQ ID NO: 123)     K           GYCTNGVCYT        YWYFDLWGRGTLVTVSS  D2-8.2  JH2                  (SEQ ID NO: 115)      (SEQ ID NO: 67) (2 = YHND) (C34D282.2A) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmt tmc tmc tmt tmt tmc tmc tmt-              tmc tmc ggt tat tgt act aac ggc gtt tgc tat act-3′    (SEQ ID NO: 124) (C34D282.2B) 5′-Ag AgT Acc cTg gcc ccA cAg gTN gAA AKA ccA AKA AKA AKA gKA-  gKA gKA gKA AKA AKA AgT ATA gcA AAc gcc gTT AgT AcA ATA-3′    (SEQ ID NO: 125)! 86 bases (5′- tat tgt act aac ggc gtt tgc tat act tmt tmt tmc tmc tmc tmc-              tmt tmt tmt tgg tmt ttc Nac ctg tgg ggc cag ggt act ct-3′    (SEQ ID NO: 126) [RC]) Design 14

Design 14 is like 9 except the D segment is mostly germline.

               1    1    2  2 2    3    3       1   5    0    5    0  3 5    0    5 YYCAK YSYYSGSYYYSDYVWGSYRYTSYDSYYYAEYFQHWGQGTLVTVSS (SEQ ID NO: 913) YYCAK ZZZZZZZZZZZDYVWGSYRZTZZZZZZZ AEZFQHWGQGTLVTVSS      (SEQ ID NO: 127)     R  D3-16.2 YYDYVWGSYRYT       AEYFQHWGQGTLVTVSS (JH1)               (SEQ ID NO: 104)    (SEQ ID NO: 66) (C34D316.2A) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmt tmc tmc tmt tmt tmc tmc tmt-              tmc tmc tmc gat tat gtc tgg ggt act tat cgt-3′     (SEQ ID NO: 128) (C34D316.2B) 5′-Ag AgT Acc cTg gcc ccA ATg cTg gAA AKA cTc Agc gKA gKA gKA-     gKA gKA gKA AKA AgT gKA Acg ATA AgT Acc ccA gAc ATA ATc-3′    (SEQ ID NO: 129) ! 86 bases (5′-gat tat gtc tgg ggt act tat cgt tmc act tmt tmc tmc tmc tmc-              tmc tmc gct gag tmt ttc cag cat tgg ggc cag ggt act ct-3′ (SEQ ID NO: 130) [RC]) Design 15

Design 15 allows some diversity in the overlap, 5 two-way flip-flops. There are only 32 overlap sequences and even if there are mismatches, they will not change the allowed diversity.

               1    1    2  2 2    3    3       1   5    0    5    0  3 5    0    5 YYCAK SYDYSSYSYYYDYVWGSYRYTSYSGDSYYAEYFQHWGQGTLVTVSS (SEQ ID NO: 914) YYCAK ZZZZZZZZZZZDZVWGZZRZTZZZZZZZZ AEZFQHWGQGTLVTVSS     (SEQ ID NO: 131)                YYDYVWGSYRYT        AEYFQH WGQGTLVTVSS                  (SEQ ID NO: 104)       (SEQ ID NO: 66) (C35D316.2A) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmt tmc tmc tmt tmt tmc tmc tmt- tmc tmc tmc gac tmt gtc tgg ggt tmc tmc cgt tmc acc t-3′ (SEQ ID NO: 132) (C35D316.2B) 5′-Ag AgT Acc cTg gcc ccA ATg cTg gAA AKA cTc Agc gKA gKA- gKA gKA gKA gKA gKA AKA ggT gKA Acg gKA gKA Acc ccA gAc AKA gTc gKA g-3′ (SEQ ID NO: 133) (5′-c tmc gac tmt gtc tgg ggt tmc tmc cgt tmc acc tmt tmc tmc- tmc tmc tmc tmc tmc gct gag tmt ttc cag cat tgg ggc cag ggt act ct-3′ (SEQ ID NO:134) [RC]) Design 16

Design 16 provides a CDR3 of 35. There are 4 two-way flip-flops in the overlap, thus 16 sequences.

               1    1    2  2 2    3    3       1   5    0    5    0  3 5    0    5 YYCAK SSSYYSYSYSGYCSGGSCYSSYYYSSYYSAEYFQGWGQGTLVTVSS (SEQ ID NO: 915) YYCAK ZZZZZZZZZZGZCZGGZCZSZZZZZZZZZ AEZFQHWGQGTLVTVSS    (SEQ ID NO: 135)     R           GYCSGGSCYS  2-25.2 AEYFQH WGQGTLVTVSS JH1                 (SEQ ID NO: 136)   (SEQ ID NO: 66) (C35D225.2A) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmt tmt tmt tmt tmt tmt tmt tmt- tmc tmc ggc tmc tgt tmc ggt ggc tmc tgc tmc tcc t-3′ (SEQ ID NO: 137) (C350225.2B) 5′-Ag AgT Acc cTg gcc ccA ATg TTg gAA AKA TTc Agc gKA KA- gKA gKA gKA gKA gKA gKA gKA gKA ggA gcA gKA gcc Acc gKA AcA gKA gcc gKA g-3′ (SEQ ID NO: 138)! 96 bases

If we add C34D225.2A and C34D225.2B to the mixture, then we get CDR3s of lengths 33, 34, and 35.

(C340225.2A) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmt tmt tmt tmt tmt tmt tmt- tmc tmc ggc tmc tgt tmc ggt ggc tmc tgc tmc tcc t-3′      (SEQ ID NO: 139) (C340225.2B) 5′-Ag AgT Acc cTg gcc ccA ATg TTg gAA AKA TTc Agc gKA gKA-    gKA gKA gKA gKA gKA gKA gKA ggA gcA gKA gcc Acc gKA AcA gKA gcc gKA g-3′  (SEQ ID NO: 140)! 93 bases Design 17

               1    1    2  2 2    3    3       1   5    0    5    0  3 5    0    5 YYCAK YSSYSYYDYVWGSYRYTSSSYSYYSYYYAEYFQGWGQGTLVTVSS (SEQ ID NO: 916) YYCAK ZZZZZZ ZDZVWGZZRZTZ ZZZZZZZZZZ AEZFQHWGQGTLVTVSS   (SEQ ID NO: 141)     R      YYDYVWGSYRYT D3-16.2   AEYFQHWGQGTLVTVSS (JH1)           (SEQ ID NO: 104)        (SEQ ID NO: 66) (C3503162A) 5′- GCA GtT taT taC tgc gct aRg tmt tmt tmt tmt tmt tmt tmc gac-    tmc gtc tgg ggt tmt tmc cgt tmt acc t-3′    (SEQ ID NO: 142) (C3503162B) 5′-Ag AgT Acc cTg gcc ccA gTg cTg gAA gKA cTc Agc gKA gKA gKA-  gKA gKA gKA gKA gKA gKA gKA gKA gKA gKA ggT AKA Acg gKA AKA Acc ccA gAc-  gKA gTc g-3′    (SEQ ID NO: 143) Design 18

               1    1    2  2 2    3    3       1   5    0    5    0  3 5    0    5 YYCAK SSYYYSSSYYDYVWGSYRYTSSYYSYSYAEYFQGWGQGTLVTVSS (SEQ ID NO: 917) YYCAK ZZZZZZZZZ ZDZVWGZZRZTZ ZZZZZZZ AEZFQHWGQGTLVTVSS    (SEQ ID NO: 144)     R         YYDYVWGSYRYT D3-16.2AEYFQHWGQGTLVTVSS (JH1)               (SEQ ID NO: 104)    (SEQ ID NO: 66) (C35D3162C) 5′- GCA|GtT|taT|taC|tgc|gct aRg tmt tmt tmt tmt tmt tmt tmc-    tmc tmc tmc gac tmc gtc tgg ggt tmc tmc cgt tmc acc t-3′ (SEQ ID NO: 145) 82 bases (C35D3162B) 5′-Ag AgT Acc cTg gcc ccA gTg cTg gAA gKA cTc Agc gKA gKA- gKA gKA gKA gKA gKA gKA gKA gKA ggT gKA Acg gKA gKA Acc ccA gAc gKA- gTc g-3′     (SEQ ID NO: 146) Design 19

               1    1    2  2 2    3    3       1   5    0    5    0  3 5    0    5 YYCAK YSGDSYSYYYYDSSGYYYSYYSSSYYSYYAEYFQGWGQGTLVTVSS (SEQ ID NO: 918) YYCAK ZZZZZZZZZ ZZDSSGZZZ ZZZZZZZZZZZ AEZFQHWGQGTLVTVSS     (SEQ ID NO: 147)     R         YYYDSSGYYY           AEYFQHWGQGTLVTVSS (JH1)                  (SEQ ID NO: 88)        (SEQ ID NO: 66)            1 1                           1     1     9 9    0 0                           0     1     4 5    0 2abcdefghijklmnopqrstuvwxyab3     0                                        ′′  Amino-acid diversity = 6.7 E 7 DNA diversity = 6.7 E 7 Stop-free = 100 Gratuitous Cys-free = 100 Free of stop and Cys = 100%

Design 19 has CDR3 of length 35. Residue 94 can be K or R. The ZZZZZZZZZ::D3-22(2^(nd) RF with six Ys as Z)::ZZZZZZZZZZZ::JH1 (with 1 Z)(SEQ ID NO:995). Error-prone PCR could be used to add more diversity.

C35D322AJH1 ! scab DNA      S   R   D   N   S   K   N   T   L   Y   L   Q   M   N   S 5′-ttc|act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|aac|agC- !              XbaI... ! !   L   R   A   E   D   T   A   V   Y   Y   C   A  K|R   |TTA|AGg|gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aRg - ! !  CDR3------ ! !  Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S  D   S   S   G  Y|S Y|S Y|S    tmc tmt tmc tmc tmt tmc tmt tmc tmc tmc tmc gac agc tcc ggc tmc tmc tmt !    Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S Y|S  A   E  Y|S  F   Q   H    tmc tmt tmc tmc tmt tmc tmt tmc tmc tmc tmc gct gaa tmc ttc caa cac ! !   W   G   Q   G   T   L   V   T   V   S   S   (SEQ ID NO: 148)    tgg ggc cag ggt act ct G GTC ACC gtc tcc agt-3′ (SEQ ID NO: 149) !                        BstEII... (C35D322AJH1_T)5′-GCA|GtT|taT|taC|tgc|gct aRg tmc tmt tmc tmc tmt- tmc tmt tmc tmc tmc tmc gac agc tcc ggc tmc tmc t-3′ (SEQ ID NO: 150) (C35D322AJH1_B) 5′-cAg AgT Acc cTg gcc ccA gTg TTg gAA gKA TTc Agc gKA-    gKA gKA gKA AKA gKA AKA gKA gKA AKA gKA AKA gKA gKA gcc ggA gcT gTc-    gKA gKA g-3′ (SEQ ID NO: 151)

ON_1, ON_2, ON_3, and ON_4 as above.

Design 20

                 1    1    2  2 2      3    3       1   5      0    5    0  3 5      0    5 YYCAK YSSYSS   YYYYDSSGYYYSSYSSYS   YYYAEYFQGWGQGTLVTVSS (SEQ ID NO: 919) YYCAK ZZZZZZ(Z)ZZ ZZDSSGZZZZZZZZZ Z(Z)ZZZ AEZFQHWGQGTLVTVSS   (SEQ ID NO: 152)     R           YYYDSSGYYY             AEYFQHWGQGTLVTVSS (JH1)               (SEQ ID NO: 88)    (SEQ ID NO: 66)            1    1                            1     1     9 9    0    0                            0     1     4 5    0    3abcdefghijklmnop q rstuvwxya4     0                                             ′ Amino-acid diversity = 6.7 E 7 DNA diversity = 6.7 E 7 Stop-free = 100 Gratuitous Cys-free = 100 Free of stop and Cy = 100%

Design 20 has CDR3s of length 33, 34, or 35. Residue 94 can be K or R. The ZZZZZZ(Z)ZZ::D3-22(2^(nd) RF with six Ys as Z)::ZZZZZZZ(Z)ZZZ::JH1(with 1 Z)(SEQ ID NO:995). PCR combining (C35D322AJH1_T), (C34D322AJH1_T), (C35D322AJH1_B), and (C34D322AJH1_B) allows length as well as sequence diversity.

(C35D322AJH1)T) (SEQ ID NO: 153) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmc tmt tmc tmc tmt tmc tmt tmc tmc tmc tmc gac agc tcc ggc tmc tmc t-3′ (C34D322AJH1)T) (SEQ ID NO: 154) 5′-GCA|GtT|taT|taC|tgc|gct aRg tmc tmc tmc tmt tmc tmt tmc tmc tmc tmc gac agc tcc ggc tmc tmc t-3′ (C350322AJH1_B) (SEQ ID NO: 920) 5′-cAg AgT Acc cTg gcc ccA gTg TTg gAA gKA TTc Agc gKA gKA gKA gKA AKA gKA AKA gKA gKA AKA gKA AKA gKA gKA gcc ggA gcT gTc-gKA gKA g-3′ (C34D322AJH1_B) (SEQ ID NO: 155) 5'-cAg AgT Acc cTg gcc ccA gTg TTg gAA gKA TTc Agc gKA gKA gKA gKA AKA gKA AKA gKA gKA AKA AKA gKA gKA gcc ggA gcT gTc gKA gKA g-3' Selection Against Stop Codons:

Because some of these libraries have NNK codons, they will have some TAG stop codons. We could remove the clones with TAG by cloning the amplified DNA into an XbaI-BstEII site between the signal sequence for a bla gene and the actual bla protein and express in Sup⁰ cells. Bla^(R) colonies do not contain TAG stops. Alternatively, we could clone the XbaI-BstEII fragments ahead of a kanamycin-resistance gene and select for Kan^(R). We would then move the XbaI-BstEII cassette into the phage library.

Also, because wobbling allows some stop codons, we can improve the library by removing the clones with stops by cloning the amplified DNA into an XbaI-BstEII site between the signal sequence for a bla gene and the actual bla protein and express in Sup⁰ cells. Bla^(R) colonies do not contain stops. Alternatively, we can clone the XbaI-BstEII fragments ahead of a kanamycin-resistance gene and select for Kan^(R). We can then move the XbaI-BstEII cassette into the phage library.

TABLE 20 Human D regions !* for TAG; @ for TAA; $ for TGA D - Amino acid sequence alignment (RF: reading frame) RF 1 RF 2 RF 3 Used in designs D1 1-1 (SEQ ID NO: 156) (SEQ ID NO: 157) (SEQ ID NO: 158) GTTGT VQLER YNWND 1-7 (SEQ ID NO: 159) (SEQ ID NO: 160) (SEQ ID NO: 161) GITGT V*LEL YNWNY 1-20 (SEQ ID NO: 159) (SEQ ID NO: 162) (SEQ ID NO: 163) GITGT V*LER YNWND 1-26 (SEQ ID NO: 164) (SEQ ID NO: 165) (SEQ ID NO: 166) GIVGAT V*WELL YSGSYY D2 2-2 (SEQ ID NOS 171 & 167) (SEQ ID NO: 70) (SEQ ID NO: 168) 1, 5, 6, 7, RIL**YQLLY GYCSSTSCYT DIVVVPAAI 2-8 (SEQ ID NOS 169 & 392) (SEQ ID NO: 115) (SEQ ID NO: 170) 20, 21, 22, RILY@WCMLY GYCTNGVCYT DIVLMVYAI 2-15 (SEQ ID NO: 171) (SEQ ID NO: 136) (SEQ ID NO: 172) 25, RIL*WW*LLL GYCSGGSCYS DIVVVVAAT 2-21 (SEQ ID NO: 173) (SEQ ID NO: 174) (SEQ ID NO: 175) SILWW$LLF AYCGGDCYS HIVVVTAI D3 3-3 (SEQ ID NO: 176) (SEQ ID NO: 177) (SEQ ID NO: 178) VLRFLEWLLY YYDFWSGYYT ITIFGVVII 3-9 (SEQ ID NO: 179) (SEQ ID NO: 180) (SEQ ID NOS 181 & 579) VLRYFDWLL@ YYDILTGYYN ITIF*LVII 3-10 (SEQ ID NO: 182) (SEQ ID NO: 81) (SEQ ID NO: 183) VLLWFGELL@ YYYGSGSYYN ITMVRGVII 3-16 (SEQ ID NO: 184) (SEQ ID NO: 104) (SEQ ID NO: 185) 8, 9, 14, 15, VL$LRLGELSLY YYDYVWGSYRYT IMITFGGVIVI 17, 18 3-22 (SEQ ID NO: 186) (SEQ ID NO: 187) (SEQ ID NO: 188) 4, 19, 20 VLL***WLLL YYYDSSGYYY ITMIVVVIT D4 4-4 (SEQ ID NO: 189) (SEQ ID NO: 192) (SEQ ID NO: 190) $LQ@L DYSNY TTVT 4-11 (SEQ ID NO: 191) (SEQ ID NO: 192) (SEQ ID NO: 193) $LQ@L DYSNY TTVT 4-17 (SEQ ID NO: 194) (SEQ ID NO: 195) (SEQ ID NO: 196) $LR@L DYGDY TTVT 4-23 (SEQ ID NO: 197) (SEQ ID NO: 198) (SEQ ID NO: 199) $LRW@L DYGGNS TTVVT D5 5-5 (SEQ ID NO: 200) (SEQ ID NO: 201) (SEQ ID NO: 202) VDTAMV WIQLWL GYSYGY 5-12 (SEQ ID NO: 203) (SEQ ID NO: 204) (SEQ ID NO: 205) VDIVATI WI*WLRL GYSGYDY 5-18 (SEQ ID NO: 206) (SEQ ID NO: 207) (SEQ ID NO: 208) VDTAMV WIQLWL GYSYGY 5-24 (SEQ ID NO: 209) (SEQ ID NO: 210) (SEQ ID NO: 211) VEMATI *RWLQL RDGYNY D6 6-6 (SEQ ID NO: 212) (SEQ ID NO: 213) (SEQ ID NO: 214) EYSSSS SIAAR V*QLV 6-13 (SEQ ID NO: 215) (SEQ ID NO: 216) (SEQ ID NO: 217) GYSSSWY GIAAAG V*QQLV 6-19 (SEQ ID NO: 218) (SEQ ID NO: 219) (SEQ ID NO: 220) GYSSGWY GIAVAG V*QWLV D7 7-27 (SEQ ID NO: 221) (SEQ ID NO: 222) (SEQ ID NO: 223) LTG @LG NWG

TABLE 3 Human JH segments JH - Amino acid sequence alignment             H3           ------            CDR3          --------             100      110               | FR4--------   Used in designs JH1   ---AEYFQH WGQGTLVTVSS 1-8,    (SEQ ID NO: 66) JH2   ---YWYFDL WGRGTLVTVSS         (SEQ ID NO: 67) JH3   -----AFDI WGQGTMVTVSS         (SEQ ID NO: 2) JH4   -----YFDY WGQGTLVTVSS         (SEQ ID NO: 1) JH5   ----NWFDP WGQGTLVTVSS         (SEQ ID NO: 68) JH6   YYYYYGMDV WGQGTTVTVSS         (SEQ ID NO: 3)       123456789

TABLE 10 DNA encoding V-5D2-8.2a-JH2 for wobbling !                                               CDR3....... !   A   E   D   T   A   V   Y   Y   C   A   K   D   I   V   L   M   |gct|gag|gaT|aCT|GCA|GtT|taT|taC|tgc|gct aag jez ezq jzz qzz ezj ! !    W   G   Q   G   T   T   V   T   V   S   S    (SEQ ID NO: 224)     tgg ggc cag ggt act acG GTC ACC gtc tcc agt-3′    (SEQ ID NO: 225) !                BstEII...

TABLE 11 Trimers that can be extracted from human D segments GTT D1-1.1.1   1 VQL D1-1.2.1   2 YNW D1-1.3.1   3 TTG D1-1.1.2   4 QLE D1-1.2.2   5 NWN D1-1.3.2   6 TGT D1-1.1.3   7 LER D1-1.2.3   8 (SEQ ID NO: 162) WND D1-1.3.3   9 GIT D1-7.1.1  10 VyL D1-7.1.2  11 * ITG D1-7.1.2  12 yLE D1-7.2.2  13 LEL D1-7.2.3  14 (SEQ ID NO: 160) WNY D1-7.3.3  15 GIV D1-26.1.1  16 VyW D1-26.2.1  17 * YSG D1-26.3.1  18 IVG D1-26.1.2  19 yWE D1-26.2.2  20 * SGS D1-26.3.2  21 VGA D1-26.1.3  22 WEL D1-26.2.3  23 GSY D1-26.3.3  24 GAT D1-26.1.4  25 ELL D1-26.2.4  26 SYY D1-26.3.4  27 RIL D2-2.1.1  28 (SEQ ID NO: 171) GYC D2-2.2.1  29 # DIV D2-2.3.1  30 ILy D2-2.1.2  31 * YCS D2-2.2.2  32 # IVV D2-2.3.2  33 Lyy D2-2.1.3  34 * CSS D2-2.2.3  35 # VVV D2-2.3.3  36 yyY D2-2.1.4  37 * SST D2-2.2.4  38 VVP D2-2.3.4  39 yYQ D2-2.2.5  40 * STS D2-2.2.5  41 VPA D2-2.3.5  42 YQL D2-2.1.6  43 TSC D2-2.2.6  44 # PAA D2-2.3.6  45 QLL D2-2.1.7  46 SCY D2-2.2.7  47 # AAI D2-2.3.7  48 LLY D2-2.1.8  49 CYT D2-2.2.8  50 # ILY D2-8.1.2  51 YCT D2-8.2.2  52 # IVL D2-8.3.2  53 LYy D2-8.1.3  54 * CTN D2-8.2.3  55 # VLM D2-8.3.3  56 YyW D2-8.1.4  57 * TNG D2-8.2.4  58 LMV D2-8.3.4  59 yWC D2-8.1.5  60 *# NGV D2-8.2.5  61 MVY D2-8.3.5  62 WCM D2-8.1.6  63 # GVC D2-8.2.6  64 # VYA D2-8.3.6  65 CML D2-8.1.7  66 # VCY D2-8.2.7  67 # YAI D2-8.3.7  68 MLY D2-8.1.8  69 LyW D2-15.1.3  70 * CSG D2-15.2.3  71 # yWW D2-15.1.4  72 * SGG D2-15.2.4  73 WWy D2-15.1.5  74 GGS D2-15.2.5  75 VVA D2-15.3.5  76 WyL D2-15.1.6  77 * GSC D2-15.2.6  78 # VAA D2-15.3.6  79 yLL D2-15.1.7  80 * AAT D2-15.3.7  81 LLL D2-15.1.8  82 CYS D2-15.2.8  83 # SIL D2-21.1.1  84 AYC D2-21.2.1  85 # HIV D2-21.3.1  86 ILW D2-21.1.2  87 YCG D2-21.2.2  88 # LWW D2-21.1.3  89 CGG D2-21.2.3  90 # WWw D2-21.1.4  91 * GGD D2-21.2.4  92 VVT D2-21.3.4  93 WwL D2-21.1.5  94 * GDC D2-21.2.5  95 # VTA D2-21.3.5  96 wLL D2-21.1.6  97 * DCY D2-21.2.6  98 # TAI D2-21.3.6  99 LLF D2-21.1.7 100 VLR D3-3.1.1 101 YYD D3-3.2.1 102 ITI D3-3.3.1 103 LRF D3-3.1.2 104 YDF D3-3.2.2 105 TIF D3-3.3.2 106 RFL D3-3.1.3 107 DFW D3-3.2.3 108 IFG D3-3.3.3 109 FLE D3-3.1.4 110 FWS D3-3.2.4 111 FGV D3-3.3.4 112 LEW D3-3.1.5 113 WSG D3-3.2.5 114 GVV D3-3.3.5 115 EWL D3-3.1.6 116 SGY D3-3.2.6 117 VVI D3-3.3.6 118 WLL D3-3.1.7 119 GYY D3-3.2.7 120 VII D3-3.3.7 121 YYT D3-3.2.8 122 LRY D3-9.1.2 123 YDI D3-9.2.2 124 RYF D3-9.1.3 125 DIL D3-9.2.3 126 IFy D3-9.3.3 127 * YFD D3-9.1.4 128 ILT D3-9.2.4 129 FyL D3-9.3.4 130 * FDW D3-9.1.5 131 LTG D3-9.2.5 132 (SEQ ID NO: 221) yLV D3-9.3.5 133 * DWL D3-9.1.6 134 TGY D3-9.2.6 135 LVI D3-9.3.6 136 LLy D3-9.1.8 137 * YYN D3-9.2.8 138 VLL D3-10.1.1 139 YYY D3-10.2.1 140 ITM D3-10.3.1 141 LLW D3-10.1.2 142 YYG D3-10.2.2 143 TMV D3-10.3.2 144 LWF D3-10.1.3 145 YGS D3-10.2.3 146 MVR D3-10.3.3 147 WFG D3-10.1.4 148 GSG D3-10.2.4 149 VRG D3-10.3.4 150 FGE D3-10.1.5 151 RGV D3-10.3.5 152 GEL D3-10.1.6 153 GVI D3-10.3.6 154 VLw D3-16.1.1 155 * IMI D3-16.3.1 156 LwL D3-16.1.2 157 * YDY D3-16.2.2 158 MIT D3-16.3.2 159 wLR D3-16.1.3 160 * DYV D3-16.2.3 161 ITF D3-16.3.3 162 LRL D3-16.1.4 163 YVW D3-16.2.4 164 TFG D3-16.3.4 165 RLG D3-16.1.5 166 VWG D3-16.2.5 167 FGG D3-16.3.5 168 LGE D3-16.1.6 169 WGS D3-16.2.6 170 GGV D3-16.3.6 171 ELS D3-16.1.8 172 SYR D3-16.2.8 173 VIV D3-16.3.8 174 LSL D3-16.1.9 175 YRY D3-16.2.9 176 IVI D3-16.3.9 177 SLY D3-16.1.10 178 RYT D3-16.2.10 179 LLw D3-22.1.2 180 * TMI D3-22.3.2 181 Lwy D3-22.1.3 182 * YDS D3-22.2.3 183 MIV D3-22.3.3 184 wyy D3-22.1.4 185 * DSS D3-22.2.4 186 yyW D3-22.1.5 187 * SSG D3-22.2.5 188 yWL D3-22.1.6 189 * VIT D3-22.3.7 190 wLQ D4-4.1.1 191 * DYS D4-4.2.1 192 TTV D4-4.3.1 193 LQy D4-4.1.2 194 * YSN D4-4.2.2 195 TVT D4-4.3.2 196 QyL D4-4.1.3 197 * SNY D4-4.2.3 198 DYG D4-17.2.1 199 LRw D4-17.1.2 200 * (SEQ ID NO: 197) YGD D4-17.2.2 201 RwL D4-17.1.3 202 * GDY D4-17.2.3 203 LRW D4-23.1.2 204 (SEQ ID NO: 197) YGG D4-23.2.2 205 TVV D4-23.3.2 206 RWy D4-23.1.3 207 * GGN D4-23.2.3 208 GNS D4-23.2.4 209 VDT D5-5.1.1 210 WIQ D5-5.2.1 211 GYS D5-5.3.1 212 DTA D5-5.1.2 213 IQL D5-5.2.2 214 YSY D5-5.3.2 215 TAM D5-5.1.3 216 QLW D5-5.2.3 217 SYG D5-5.3.3 218 AMV D5-5.1.4 219 LWL D5-5.2.4 220 YGY D5-5.3.4 221 VDI D5-12.1.1 222 WIy D5-12.2.1 223 * IyW D5-12.2.2 224 * IVA D5-12.1.3 225 VAT D5-12.1.4 226 WLR D5-12.2.4 227 GYD D5-12.3.4 228 ATI D5-12.1.5 229 VEM D5-24.1.1 230 yRW D5-24.2.1 231 * RDG D5-24.3.1 232 EMA D5-24.1.2 233 RWL D5-24.2.2 234 DGY D5-24.3.2 235 MAT D5-24.1.3 236 WLQ D5-24.2.3 237 GYN D5-24.3.3 238 LQL D5-24.2.4 239 YNY D5-24.3.4 240 EYS D6-6.1.1 241 SIA D6-6.2.1 242 VyQ D6-6.3.1 243 * YSS D6-6.1.2 244 IAA D6-6.2.2 245 yQL D6-6.3.2 246 * SSS D6-6.1.3 247 AAR D6-6.2.3 248 QLV D6-6.3.3 249 (SEQ ID NO: 214) GIA D6-13.2.1 250 yQQ D6-13.3.2 251 * AAA D6-13.2.3 252 QQL D6-13.3.3 253 SSW D6-13.1.4 254 AAG D6-13.2.4 255 SWY D6-13.1.5 256 IAV D6-19.2.2 257 yQW D6-19.3.2 258 * AVA D6-19.2.3 259 QWL D6-19.3.3 260 SGW D6-19.1.4 261 VAG D6-19.2.4 262 WLV D6-19.3.4 263 GWY D6-19.1.5 264 yLG D7-27.2.1 265 * NWG D7-27.3.1 266 (SEQ ID NO: 223)

In Tables 11-14, the use of a lower case letter in an amino acid sequence indicates that a stop codon was changed to the residue listed as the lower case letter. For example, in the amino acid sequence “yLE”, a Tyr residue was introduced in place of a stop codon.

TABLE 12 Distinct tetramers that can be extracted from human D segments GTTG D1-1.1.1 (SEQ ID NO: 257) 1 VQLE D1-1.2.1 (SEQ ID NO: 258) 2 YNWN D1-1.3.1 (SEQ ID NO: 259) 3 TTGT D1-1.1.2 (SEQ ID NO: 263) 4 QLER D1-1.2.2 (SEQ ID NO: 264) 5 NWND D1-1.3.2 (SEQ ID NO: 265) 6 GITG D1-7.1.1 (SEQ ID NO: 266) 7 VyLE D1-7.2.1 (SEQ ID NO: 267) 8 ITGT D1-7.1.2 (SEQ ID NO: 271) 9 yLEL D1-7.2.2 (SEQ ID NO: 272) 10 NWNY D1-7.3.2 (SEQ ID NO: 273) 11 yLER D1-20.2.2 (SEQ ID NO: 275) 12 GIVG D1-26.1.1 (SEQ ID NO: 276) 13 VyWE D1-26.2.1 (SEQ ID NO: 277) 14 YSGS D1-26.3.1 (SEQ ID NO: 278) 15 IVGA D1-26.1.2 (SEQ ID NO: 285) 16 yWEL D1-26.2.2 (SEQ ID NO: 286) 17 SGSY D1-26.3.2 (SEQ ID NO: 287) 18 VGAT D1-26.1.3 (SEQ ID NO: 291) 19 WELL D1-26.2.3 (SEQ ID NO: 292) 20 GSYY D1-26.3.3 (SEQ ID NO: 293) 21 RILy D2-2.1.1 (SEQ ID NO: 294) 22 GYCS D2-2.2.1 (SEQ ID NO: 295) 23 DIVV D2-2.3.1 (SEQ ID NO: 296) 24 ILyy D2-2.1.2 (SEQ ID NO: 303) 25 YCSS D2-2.2.2 (SEQ ID NO: 304) 26 IVVV D2-2.3.2 (SEQ ID NO: 305) 27 LyyY D2-2.1.3 (SEQ ID NO: 312) 28 CSST D2-2.2.3 (SEQ ID NO: 313) 29 VVVP D2-2.3.3 (SEQ ID NO: 314) 30 yyYQ D2-2.1.4 (SEQ ID NO: 321) 31 SSTS D2-2.2.4 (SEQ ID NO: 322) 32 VVPA D2-2.3.4 (SEQ ID NO: 323) 33 yYQL D2-2.1.5 (SEQ ID NO: 330) 34 STSC D2-2.2.5 (SEQ ID NO: 331) 35 VPAA D2-2.3.5 (SEQ ID NO: 332) 36 YQLL D2-2.1.6 (SEQ ID NO: 338) 37 TSCY D2-2.2.6 (SEQ ID NO: 339) 38 PAAI D2-2.3.6 (SEQ ID NO: 340) 39 QLLY D2-2.1.7 (SEQ ID NO: 343) 40 SCYT D2-2.2.7 (SEQ ID NO: 344) 41 RILY D2-8.1.1 (SEQ ID NO: 345) 42 GYCT D2-8.2.1 (SEQ ID NO: 346) 43 DIVL D2-8.3.1 (SEQ ID NO: 347) 44 ILYy D2-8.1.2 (SEQ ID NO: 354) 45 YCTN D2-8.2.2 (SEQ ID NO: 355) 46 IVLM D2-8.3.2 (SEQ ID NO: 356) 47 LYyW D2-8.1.3 (SEQ ID NO: 363) 48 CTNG D2-8.2.3 (SEQ ID NO: 364) 49 VLMV D2-8.3.3 (SEQ ID NO: 365) 50 YyWC D2-8.1.4 (SEQ ID NO: 372) 51 TNGV D2-8.2.4 (SEQ ID NO: 373) 52 LMVY D2-8.3.4 (SEQ ID NO: 374) 53 yWCM D2-8.1.5 (SEQ ID NO: 381) 54 NGVC D2-8.2.5 (SEQ ID NO: 382) 55 MVYA D2-8.3.5 (SEQ ID NO: 383) 56 WCML D2-8.1.6 (SEQ ID NO: 389) 57 GVCY D2-8.2.6 (SEQ ID NO: 390) 58 VYAI D2-8.3.6 (SEQ ID NO: 391) 59 CMLY D2-8.1.7 (SEQ ID NO: 394) 60 VCYT D2-8.2.7 (SEQ ID NO: 395) 61 ILyW D2-15.1.2 (SEQ ID NO: 401) 62 YCSG D2-15.2.2 (SEQ ID NO: 402) 63 LyWW D2-15.1.3 (SEQ ID NO: 409) 64 CSGG D2-15.2.3 (SEQ ID NO: 410) 65 VVVV D2-15.3.3 (SEQ ID NO: 411) 66 yWWy D2-15.1.4 (SEQ ID NO: 418) 67 SGGS D2-15.2.4 (SEQ ID NO: 419) 68 VVVA D2-15.3.4 (SEQ ID NO: 420) 69 WWyL D2-15.1.5 (SEQ ID NO: 427) 70 GGSC D2-15.2.5 (SEQ ID NO: 428) 71 VVAA D2-15.3.5 (SEQ ID NO: 429) 72 WyLL D2-15.1.6 (SEQ ID NO: 435) 73 GSCY D2-15.2.6 (SEQ ID NO: 436) 74 VAAT D2-15.3.6 (SEQ ID NO: 437) 75 yLLL D2-15.1.7 (SEQ ID NO: 440) 76 SCYS D2-15.2.7 (SEQ ID NO: 441) 77 SILW D2-21.1.1 (SEQ ID NO: 442) 78 AYCG D2-21.2.1 (SEQ ID NO: 443) 79 HIVV D2-21.3.1 (SEQ ID NO: 444) 80 ILWW D2-21.1.2 (SEQ ID NO: 451) 81 YCGG D2-21.2.2 (SEQ ID NO: 452) 82 LWWw D2-21.1.3 (SEQ ID NO: 459) 83 CGGD D2-21.2.3 (SEQ ID NO: 460) 84 VVVT D2-21.3.3 (SEQ ID NO: 461) 85 WWwL D2-21.1.4 (SEQ ID NO: 468) 86 GGDC D2-21.2.4 (SEQ ID NO: 469) 87 VVTA D2-21.3.4 (SEQ ID NO: 470) 88 WwLL D2-21.1.5 (SEQ ID NO: 476) 89 GDCY D2-21.2.5 (SEQ ID NO: 477) 90 VTAI D2-21.3.5 (SEQ ID NO: 478) 91 wLLF D2-21.1.6 (SEQ ID NO: 481) 92 DCYS D2-21.2.6 (SEQ ID NO: 482) 93 VLRF D3-3.1.1 (SEQ ID NO: 483) 94 YYDF D3-3.2.1 (SEQ ID NO: 484) 95 ITIF D3-3.3.1 (SEQ ID NO: 485) 96 LRFL D3-3.1.2 (SEQ ID NO: 492) 97 YDFW D3-3.2.2 (SEQ ID NO: 493) 98 TIFG D3-3.3.2 (SEQ ID NO: 494) 99 RFLE D3-3.1.3 (SEQ ID NO: 501) 100 DFWS D3-3.2.3 (SEQ ID NO: 502) 101 IFGV D3-3.3.3 (SEQ ID NO: 503) 102 FLEW D3-3.1.4 (SEQ ID NO: 510) 103 FWSG D3-3.2.4 (SEQ ID NO: 511) 104 FGVV D3-3.3.4 (SEQ ID NO: 512) 105 LEWL D3-3.1.5 (SEQ ID NO: 519) 106 WSGY D3-3.2.5 (SEQ ID NO: 520) 107 GVVI D3-3.3.5 (SEQ ID NO: 521) 108 EWLL D3-3.1.6 (SEQ ID NO: 527) 109 SGYY D3-3.2.6 (SEQ ID NO: 528) 110 VVII D3-3.3.6 (SEQ ID NO: 529) 111 WLLY D3-3.1.7 (SEQ ID NO: 532) 112 GYYT D3-3.2.7 (SEQ ID NO: 533) 113 VLRY D3-9.1.1 (SEQ ID NO: 534) 114 YYDI D3-9.2.1 (SEQ ID NO: 535) 115 LRYF D3-9.1.2 (SEQ ID NO: 542) 116 YDIL D3-9.2.2 (SEQ ID NO: 543) 117 TIFy D3-9.3.2 (SEQ ID NO: 544) 118 RYFD D3-9.1.3 (SEQ ID NO: 551) 119 DILT D3-9.2.3 (SEQ ID NO: 552) 120 IFyL D3-9.3.3 (SEQ ID NO: 553) 121 YFDW D3-9.1.4 (SEQ ID NO: 560) 122 ILTG D3-9.2.4 (SEQ ID NO: 561) 123 FyLV D3-9.3.4 (SEQ ID NO: 562) 124 FDWL D3-9.1.5 (SEQ ID NO: 569) 125 LTGY D3-9.2.5 (SEQ ID NO: 570) 126 yLVI D3-9.3.5 (SEQ ID NO: 571) 127 DWLL D3-9.1.6 (SEQ ID NO: 577) 128 TGYY D3-9.2.6 (SEQ ID NO: 578) 129 LVII D3-9.3.6 (SEQ ID NO: 579) 130 WLLy D3-9.1.7 (SEQ ID NO: 582) 131 GYYN D3-9.2.7 (SEQ ID NO: 583) 132 VLLW D3-10.1.1 (SEQ ID NO: 584) 133 YYYG D3-10.2.1 (SEQ ID NO: 585) 134 ITMV D3-10.3.1 (SEQ ID NO: 586) 135 LLWF D3-10.1.2 (SEQ ID NO: 593) 136 YYGS D3-10.2.2 (SEQ ID NO: 594) 137 TMVR D3-10.3.2 (SEQ ID NO: 595) 138 LWFG D3-10.1.3 (SEQ ID NO: 602) 139 YGSG D3-10.2.3 (SEQ ID NO: 603) 140 MVRG D3-10.3.3 (SEQ ID NO: 604) 141 WFGE D3-10.1.4 (SEQ ID NO: 611) 142 GSGS D3-10.2.4 (SEQ ID NO: 612) 143 VRGV D3-10.3.4 (SEQ ID NO: 613) 144 FGEL D3-10.1.5 (SEQ ID NO: 620) 145 RGVI D3-10.3.5 (SEQ ID NO: 621) 146 GELL D3-10.1.6 (SEQ ID NO: 626) 147 GVII D3-10.3.6 (SEQ ID NO: 627) 148 ELLy D3-10.1.7 (SEQ ID NO: 630) 149 SYYN D3-10.2.7 (SEQ ID NO: 631) 150 VLwL D3-16.1.1 (SEQ ID NO: 632) 151 YYDY D3-16.2.1 (SEQ ID NO: 633) 152 IMIT D3-16.3.1 (SEQ ID NO: 634) 153 LwLR D3-16.1.2 (SEQ ID NO: 641) 154 YDYV D3-16.2.2 (SEQ ID NO: 642) 155 MITF D3-16.3.2 (SEQ ID NO: 643) 156 wLRL D3-16.1.3 (SEQ ID NO: 650) 157 DYVW D3-16.2.3 (SEQ ID NO: 651) 158 ITFG D3-16.3.3 (SEQ ID NO: 652) 159 LRLG D3-16.1.4 (SEQ ID NO: 659) 160 YVWG D3-16.2.4 (SEQ ID NO: 660) 161 TFGG D3-16.3.4 (SEQ ID NO: 661) 162 RLGE D3-16.1.5 (SEQ ID NO: 668) 163 VWGS D3-16.2.5 (SEQ ID NO: 669) 164 FGGV D3-16.3.5 (SEQ ID NO: 670) 165 LGEL D3-16.1.6 (SEQ ID NO: 677) 166 WGSY D3-16.2.6 (SEQ ID NO: 678) 167 GGVI D3-16.3.6 (SEQ ID NO: 679) 168 GELS D3-16.1.7 (SEQ ID NO: 686) 169 GSYR D3-16.2.7 (SEQ ID NO: 687) 170 GVIV D3-16.3.7 (SEQ ID NO: 688) 171 ELSL D3-16.1.8 (SEQ ID NO: 694) 172 SYRY D3-16.2.8 (SEQ ID NO: 695) 173 VIVI D3-16.3.8 (SEQ ID NO: 696) 174 LSLY D3-16.1.9 (SEQ ID NO: 699) 175 YRYT D3-16.2.9 (SEQ ID NO: 700) 176 VLLw D3-22.1.1 (SEQ ID NO: 701) 177 YYYD D3-22.2.1 (SEQ ID NO: 702) 178 ITMI D3-22.3.1 (SEQ ID NO: 703) 179 LLwy D3-22.1.2 (SEQ ID NO: 710) 180 YYDS D3-22.2.2 (SEQ ID NO: 711) 181 TMIV D3-22.3.2 (SEQ ID NO: 712) 182 Lwyy D3-22.1.3 (SEQ ID NO: 719) 183 YDSS D3-22.2.3 (SEQ ID NO: 720) 184 MIVV D3-22.3.3 (SEQ ID NO: 721) 185 wyyW D3-22.1.4 (SEQ ID NO: 728) 186 DSSG D3-22.2.4 (SEQ ID NO: 729) 187 yyWL D3-22.1.5 (SEQ ID NO: 736) 188 SSGY D3-22.2.5 (SEQ ID NO: 737) 189 VVVI D3-22.3.5 (SEQ ID NO: 738) 190 yWLL D3-22.1.6 (SEQ ID NO: 744) 191 VVIT D3-22.3.6 (SEQ ID NO: 745) 192 WLLL D3-22.1.7 (SEQ ID NO: 748) 193 GYYY D3-22.2.7 (SEQ ID NO: 749) 194 wLQy D4-4.1.1 (SEQ ID NO: 750) 195 DYSN D4-4.2.1 (SEQ ID NO: 751) 196 TTVT D4-4.3.1 (SEQ ID NO: 752) 197 LQyL D4-4.1.2 (SEQ ID NO: 755) 198 YSNY D4-4.2.2 (SEQ ID NO: 756) 199 wLRw D4-17.1.1 (SEQ ID NO: 757) 200 DYGD D4-17.2.1 (SEQ ID NO: 758) 201 LRwL D4-17.1.2 (SEQ ID NO: 761) 202 YGDY D4-17.2.2 (SEQ ID NO: 762) 203 wLRW D4-23.1.1 (SEQ ID NO: 763) 204 DYGG D4-23.2.1 (SEQ ID NO: 764) 205 TTVV D4-23.3.1 (SEQ ID NO: 765) 206 LRWy D4-23.1.2 (SEQ ID NO: 771) 207 YGGN D4-23.2.2 (SEQ ID NO: 772) 208 TVVT D4-23.3.2 (SEQ ID NO: 773) 209 RWyL D4-23.1.3 (SEQ ID NO: 776) 210 GGNS D4-23.2.3 (SEQ ID NO: 777) 211 VDTA D5-5.1.1 (SEQ ID NO: 778) 212 WIQL D5-5.2.1 (SEQ ID NO: 779) 213 GYSY D5-5.3.1 (SEQ ID NO: 780) 214 DTAM D5-5.1.2 (SEQ ID NO: 787) 215 IQLW D5-5.2.2 (SEQ ID NO: 788) 216 YSYG D5-5.3.2 (SEQ ID NO: 789) 217 TAMV D5-5.1.3 (SEQ ID NO: 793) 218 QLWL D5-5.2.3 (SEQ ID NO: 794) 219 SYGY D5-5.3.3 (SEQ ID NO: 795) 220 VDIV D5-12.1.1 (SEQ ID NO: 796) 221 WIyW D5-12.2.1 (SEQ ID NO: 797) 222 GYSG D5-12.3.1 (SEQ ID NO: 798) 223 DIVA D5-12.1.2 (SEQ ID NO: 805) 224 IyWL D5-12.2.2 (SEQ ID NO: 806) 225 YSGY D5-12.3.2 (SEQ ID NO: 807) 226 IVAT D5-12.1.3 (SEQ ID NO: 814) 227 yWLR D5-12.2.3 (SEQ ID NO: 815) 228 SGYD D5-12.3.3 (SEQ ID NO: 816) 229 VATI D5-12.1.4 (SEQ ID NO: 820) 230 WLRL D5-12.2.4 (SEQ ID NO: 821) 231 GYDY D5-12.3.4 (SEQ ID NO: 822) 232 VEMA D5-24.1.1 (SEQ ID NO: 823) 233 yRWL D5-24.2.1 (SEQ ID NO: 824) 234 RDGY D5-24.3.1 (SEQ ID NO: 825) 235 EMAT D5-24.1.2 (SEQ ID NO: 832) 236 RWLQ D5-24.2.2 (SEQ ID NO: 833) 237 DGYN D5-24.3.2 (SEQ ID NO: 834) 238 MATI D5-24.1.3 (SEQ ID NO: 838) 239 WLQL D5-24.2.3 (SEQ ID NO: 839) 240 GYNY D5-24.3.3 (SEQ ID NO: 840) 241 EYSS D6-6.1.1 (SEQ ID NO: 841) 242 SIAA D6-6.2.1 (SEQ ID NO: 842) 243 VyQL D6-6.3.1 (SEQ ID NO: 843) 244 YSSS D6-6.1.2 (SEQ ID NO: 848) 245 IAAR D6-6.2.2 (SEQ ID NO: 849) 246 yQLV D6-6.3.2 (SEQ ID NO: 850) 247 SSSS D6-6.1.3 (SEQ ID NO: 852) 248 GYSS D6-13.1.1 (SEQ ID NO: 853) 249 GIAA D6-13.2.1 (SEQ ID NO: 854) 250 VyQQ D6-13.3.1 (SEQ ID NO: 855) 251 IAAA D6-13.2.2 (SEQ ID NO: 862) 252 yQQL D6-13.3.2 (SEQ ID NO: 863) 253 SSSW D6-13.1.3 (SEQ ID NO: 868) 254 AAAG D6-13.2.3 (SEQ ID NO: 869) 255 QQLV D6-13.3.3 (SEQ ID NO: 870) 256 SSWY D6-13.1.4 (SEQ ID NO: 872) 257 GIAV D6-19.2.1 (SEQ ID NO: 873) 258 VyQW D6-19.3.1 (SEQ ID NO: 874) 259 YSSG D6-19.1.2 (SEQ ID NO: 881) 260 IAVA D6-19.2.2 (SEQ ID NO: 882) 261 yQWL D6-19.3.2 (SEQ ID NO: 883) 262 SSGW D6-19.1.3 (SEQ ID NO: 888) 263 AVAG D6-19.2.3 (SEQ ID NO: 889) 264 QWLV D6-19.3.3 (SEQ ID NO: 890) 265 SGWY D6-19.1.4 (SEQ ID NO: 941) 266

TABLE 13 Pentamers that can be extracted from human D segments GTTGT D1-1.1.1 (SEQ ID NO: 260) 1 VQLER D1-1.2.1 (SEQ ID NO: 261) 2 YNWND D1-1.3.1 (SEQ ID NO: 262) 3 GITGT D1-7.1.1 (SEQ ID NO: 268) 4 VyLEL D1-7.2.1 (SEQ ID NO: 269) 5 YNWNY D1-7.3.1 (SEQ ID NO: 270) 6 VyLER D1-20.2.1 (SEQ ID NO: 274) 7 GIVGA D1-26.1.1 (SEQ ID NO: 279) 8 VyWEL D1-26.2.1 (SEQ ID NO: 280) 9 YSGSY D1-26.3.1 (SEQ ID NO: 281) 10 IVGAT D1-26.1.2 (SEQ ID NO: 288) 11 yWELL D1-26.2.2 (SEQ ID NO: 289) 12 SGSYY D1-26.3.2 (SEQ ID NO: 290) 13 RILyy D2-2.1.1 (SEQ ID NO: 297) 14 GYCSS D2-2.2.1 (SEQ ID NO: 298) 15 DIVVV D2-2.3.1 (SEQ ID NO: 299) 16 ILyyY D2-2.1.2 (SEQ ID NO: 306) 17 YCSST D2-2.2.2 (SEQ ID NO: 307) 18 IVVVP D2-2.3.2 (SEQ ID NO: 308) 19 LyyYQ D2-2.1.3 (SEQ ID NO: 315) 20 CSSTS D2-2.2.3 (SEQ ID NO: 316) 21 VVVPA D2-2.3.3 (SEQ ID NO: 317) 22 yyYQL D2-2.1.4 (SEQ ID NO: 324) 23 SSTSC D2-2.2.4 (SEQ ID NO: 325) 24 VVPAA D2-2.3.4 (SEQ ID NO: 326) 25 yYQLL D2-2.1.5 (SEQ ID NO: 333) 26 STSCY D2-2.2.5 (SEQ ID NO: 334) 27 VPAAI D2-2.3.5 (SEQ ID NO: 335) 28 YQLLY D2-2.1.6 (SEQ ID NO: 341) 29 TSCYT D2-2.2.6 (SEQ ID NO: 342) 30 RILYy D2-8.1.1 (SEQ ID NO: 348) 31 GYCTN D2-8.2.1 (SEQ ID NO: 349) 32 DIVLM D2-8.3.1 (SEQ ID NO: 350) 33 ILYyW D2-8.1.2 (SEQ ID NO: 357) 34 YCTNG D2-8.2.2 (SEQ ID NO: 358) 35 IVLMV D2-8.3.2 (SEQ ID NO: 359) 36 LYyWC D2-8.1.3 (SEQ ID NO: 366) 37 CTNGV D2-8.2.3 (SEQ ID NO: 367) 38 VLMVY D2-8.3.3 (SEQ ID NO: 368) 39 YyWCM D2-8.1.4 (SEQ ID NO: 375) 40 TNGVC D2-8.2.4 (SEQ ID NO: 376) 41 LMVYA D2-8.3.4 (SEQ ID NO: 377) 42 yWCML D2-8.1.5 (SEQ ID NO: 384) 43 NGVCY D2-8.2.5 (SEQ ID NO: 385) 44 MVYAI D2-8.3.5 (SEQ ID NO: 386) 45 WCMLY D2-8.1.6 (SEQ ID NO: 392) 46 GVCYT D2-8.2.6 (SEQ ID NO: 393) 47 RILyW D2-15.1.1 (SEQ ID NO: 396) 48 GYCSG D2-15.2.1 (SEQ ID NO: 397) 49 ILyWW D2-15.1.2 (SEQ ID NO: 403) 50 YCSGG D2-15.2.2 (SEQ ID NO: 404) 51 IVVVV D2-15.3.2 (SEQ ID NO: 405) 52 LyWWy D2-15.1.3 (SEQ ID NO: 412) 53 CSGGS D2-15.2.3 (SEQ ID NO: 413) 54 VVVVA D2-15.3.3 (SEQ ID NO: 414) 55 yWWyL D2-15.1.4 (SEQ ID NO: 421) 56 SGGSC D2-15.2.4 (SEQ ID NO: 422) 57 VVVAA D2-15.3.4 (SEQ ID NO: 423) 58 WWyLL D2-15.1.5 (SEQ ID NO: 430) 59 GGSCY D2-15.2.5 (SEQ ID NO: 431) 60 VVAAT D2-15.3.5 (SEQ ID NO: 432) 61 WyLLL D2-15.1.6 (SEQ ID NO: 438) 62 GSCYS D2-15.2.6 (SEQ ID NO: 439) 63 SILWW D2-21.1.1 (SEQ ID NO: 445) 64 AYCGG D2-21.2.1 (SEQ ID NO: 446) 65 HIVVV D2-21.3.1 (SEQ ID NO: 447) 66 ILWWw D2-21.1.2 (SEQ ID NO: 453) 67 YCGGD D2-21.2.2 (SEQ ID NO: 454) 68 IVVVT D2-21.3.2 (SEQ ID NO: 455) 69 LWWwL D2-21.1.3 (SEQ ID NO: 462) 70 CGGDC D2-21.2.3 (SEQ ID NO: 463) 71 VVVTA D2-21.3.3 (SEQ ID NO: 464) 72 WWwLL D2-21.1.4 (SEQ ID NO: 471) 73 GGDCY D2-21.2.4 (SEQ ID NO: 472) 74 VVTAI D2-21.3.4 (SEQ ID NO: 473) 75 WwLLF D2-21.1.5 (SEQ ID NO: 479) 76 GDCYS D2-21.2.5 (SEQ ID NO: 480) 77 VLRFL D3-3.1.1 (SEQ ID NO: 486) 78 YYDFW D3-3.2.1 (SEQ ID NO: 487) 79 ITIFG D3-3.3.1 (SEQ ID NO: 488) 80 LRFLE D3-3.1.2 (SEQ ID NO: 495) 81 YDFWS D3-3.2.2 (SEQ ID NO: 496) 82 TIFGV D3-3.3.2 (SEQ ID NO: 497) 83 RFLEW D3-3.1.3 (SEQ ID NO: 504) 84 DFWSG D3-3.2.3 (SEQ ID NO: 505) 85 IFGVV D3-3.3.3 (SEQ ID NO: 506) 86 FLEWL D3-3.1.4 (SEQ ID NO: 513) 87 FWSGY D3-3.2.4 (SEQ ID NO: 514) 88 FGVVI D3-3.3.4 (SEQ ID NO: 515) 89 LEWLL D3-3.1.5 (SEQ ID NO: 522) 90 WSGYY D3-3.2.5 (SEQ ID NO: 523) 91 GVVII D3-3.3.5 (SEQ ID NO: 524) 92 EWLLY D3-3.1.6 (SEQ ID NO: 530) 93 SGYYT D3-3.2.6 (SEQ ID NO: 531) 94 VLRYF D3-9.1.1 (SEQ ID NO: 536) 95 YYDIL D3-9.2.1 (SEQ ID NO: 537) 96 ITIFy D3-9.3.1 (SEQ ID NO: 538) 97 LRYFD D3-9.1.2 (SEQ ID NO: 545) 98 YDILT D3-9.2.2 (SEQ ID NO: 546) 99 TIFyL D3-9.3.2 (SEQ ID NO: 547) 100 RYFDW D3-9.1.3 (SEQ ID NO: 554) 101 DILTG D3-9.2.3 (SEQ ID NO: 555) 102 IFyLV D3-9.3.3 (SEQ ID NO: 556) 103 YFDWL D3-9.1.4 (SEQ ID NO: 563) 104 ILTGY D3-9.2.4 (SEQ ID NO: 564) 105 FyLVI D3-9.3.4 (SEQ ID NO: 565) 106 FDWLL D3-9.1.5 (SEQ ID NO: 572) 107 LTGYY D3-9.2.5 (SEQ ID NO: 573) 108 yLVII D3-9.3.5 (SEQ ID NO: 574) 109 DWLLy D3-9.1.6 (SEQ ID NO: 580) 110 TGYYN D3-9.2.6 (SEQ ID NO: 581) 111 VLLWF D3-10.1.1 (SEQ ID NO: 587) 112 YYYGS D3-10.2.1 (SEQ ID NO: 588) 113 ITMVR D3-10.3.1 (SEQ ID NO: 589) 114 LLWFG D3-10.1.2 (SEQ ID NO: 596) 115 YYGSG D3-10.2.2 (SEQ ID NO: 597) 116 TMVRG D3-10.3.2 (SEQ ID NO: 598) 117 LWFGE D3-10.1.3 (SEQ ID NO: 605) 118 YGSGS D3-10.2.3 (SEQ ID NO: 606) 119 MVRGV D3-10.3.3 (SEQ ID NO: 607) 120 WFGEL D3-10.1.4 (SEQ ID NO: 614) 121 GSGSY D3-10.2.4 (SEQ ID NO: 615) 122 VRGVI D3-10.3.4 (SEQ ID NO: 616) 123 FGELL D3-10.1.5 (SEQ ID NO: 622) 124 RGVII D3-10.3.5 (SEQ ID NO: 623) 125 GELLy D3-10.1.6 (SEQ ID NO: 628) 126 GSYYN D3-10.2.6 (SEQ ID NO: 629) 127 VLwLR D3-16.1.1 (SEQ ID NO: 635) 128 YYDYV D3-16.2.1 (SEQ ID NO: 636) 129 IMITF D3-16.3.1 (SEQ ID NO: 637) 130 LwLRL D3-16.1.2 (SEQ ID NO: 644) 131 YDYVW D3-16.2.2 (SEQ ID NO: 645) 132 MITFG D3-16.3.2 (SEQ ID NO: 646) 133 wLRLG D3-16.1.3 (SEQ ID NO: 653) 134 DYVWG D3-16.2.3 (SEQ ID NO: 654) 135 ITFGG D3-16.3.3 (SEQ ID NO: 655) 136 LRLGE D3-16.1.4 (SEQ ID NO: 662) 137 YVWGS D3-16.2.4 (SEQ ID NO: 663) 138 TFGGV D3-16.3.4 (SEQ ID NO: 664) 139 RLGEL D3-16.1.5 (SEQ ID NO: 671) 140 VWGSY D3-16.2.5 (SEQ ID NO: 672) 141 FGGVI D3-16.3.5 (SEQ ID NO: 673) 142 LGELS D3-16.1.6 (SEQ ID NO: 680) 143 WGSYR D3-16.2.6 (SEQ ID NO: 681) 144 GGVIV D3-16.3.6 (SEQ ID NO: 682) 145 GELSL D3-16.1.7 (SEQ ID NO: 689) 146 GSYRY D3-16.2.7 (SEQ ID NO: 690) 147 GVIVI D3-16.3.7 (SEQ ID NO: 691) 148 ELSLY D3-16.1.8 (SEQ ID NO: 697) 149 SYRYT D3-16.2.8 (SEQ ID NO: 698) 150 VLLwy D3-22.1.1 (SEQ ID NO: 704) 151 YYYDS D3-22.2.1 (SEQ ID NO: 705) 152 ITMIV D3-22.3.1 (SEQ ID NO: 706) 153 LLwyy D3-22.1.2 (SEQ ID NO: 713) 154 YYDSS D3-22.2.2 (SEQ ID NO: 714) 155 TMIVV D3-22.3.2 (SEQ ID NO: 715) 156 LwyyW D3-22.1.3 (SEQ ID NO: 722) 157 YDSSG D3-22.2.3 (SEQ ID NO: 723) 158 MIVVV D3-22.3.3 (SEQ ID NO: 724) 159 wyyWL D3-22.1.4 (SEQ ID NO: 730) 160 DSSGY D3-22.2.4 (SEQ ID NO: 731) 161 IVVVI D3-22.3.4 (SEQ ID NO: 732) 162 yyWLL D3-22.1.5 (SEQ ID NO: 739) 163 SSGYY D3-22.2.5 (SEQ ID NO: 740) 164 VVVIT D3-22.3.5 (SEQ ID NO: 741) 165 yWLLL D3-22.1.6 (SEQ ID NO: 746) 166 SGYYY D3-22.2.6 (SEQ ID NO: 747) 167 wLQyL D4-4.1.1 (SEQ ID NO: 753) 168 DYSNY D4-4.2.1 (SEQ ID NO: 754) 169 wLRwL D4-17.1.1 (SEQ ID NO: 759) 170 DYGDY D4-17.2.1 (SEQ ID NO: 760) 171 wLRWy D4-23.1.1 (SEQ ID NO: 766) 172 DYGGN D4-23.2.1 (SEQ ID NO: 767) 173 TTVVT D4-23.3.1 (SEQ ID NO: 768) 174 LRWyL D4-23.1.2 (SEQ ID NO: 774) 175 YGGNS D4-23.2.2 (SEQ ID NO: 775) 176 VDTAM D5-5.1.1 (SEQ ID NO: 781) 177 WIQLW D5-5.2.1 (SEQ ID NO: 782) 178 GYSYG D5-5.3.1 (SEQ ID NO: 783) 179 DTAMV D5-5.1.2 (SEQ ID NO: 790) 180 IQLWL D5-5.2.2 (SEQ ID NO: 791) 181 YSYGY D5-5.3.2 (SEQ ID NO: 792) 182 VDIVA D5-12.1.1 (SEQ ID NO: 799) 183 WIyWL D5-12.2.1 (SEQ ID NO: 800) 184 GYSGY D5-12.3.1 (SEQ ID NO: 801) 185 DIVAT D5-12.1.2 (SEQ ID NO: 808) 186 IyWLR D5-12.2.2 (SEQ ID NO: 809) 187 YSGYD D5-12.3.2 (SEQ ID NO: 810) 188 IVATI D5-12.1.3 (SEQ ID NO: 817) 189 yWLRL D5-12.2.3 (SEQ ID NO: 818) 190 SGYDY D5-12.3.3 (SEQ ID NO: 819) 191 VEMAT D5-24.1.1 (SEQ ID NO: 826) 192 yRWLQ D5-24.2.1 (SEQ ID NO: 827) 193 RDGYN D5-24.3.1 (SEQ ID NO: 828) 194 EMATI D5-24.1.2 (SEQ ID NO: 835) 195 RWLQL D5-24.2.2 (SEQ ID NO: 836) 196 DGYNY D5-24.3.2 (SEQ ID NO: 837) 197 EYSSS D6-6.1.1 (SEQ ID NO: 844) 198 SIAAR D6-6.2.1 (SEQ ID NO: 845) 199 VyQLV D6-6.3.1 (SEQ ID NO: 846) 200 YSSSS D6-6.1.2 (SEQ ID NO: 851) 201 GYSSS D6-13.1.1 (SEQ ID NO: 856) 202 GIAAA D6-13.2.1 (SEQ ID NO: 857) 203 VyQQL D6-13.3.1 (SEQ ID NO: 858) 204 YSSSW D6-13.1.2 (SEQ ID NO: 864) 205 IAAAG D6-13.2.2 (SEQ ID NO: 865) 206 yQQLV D6-13.3.2 (SEQ ID NO: 866) 207 SSSWY D6-13.1.3 (SEQ ID NO: 871) 208 GYSSG D6-19.1.1 (SEQ ID NO: 875) 209 GIAVA D6-19.2.1 (SEQ ID NO: 876) 210 VyQWL D6-19.3.1 (SEQ ID NO: 877) 211 YSSGW D6-19.1.2 (SEQ ID NO: 884) 212 IAVAG D6-19.2.2 (SEQ ID NO: 885) 213 yQWLV D6-19.3.2 (SEQ ID NO: 886) 214 SSGWY D6-19.1.3 (SEQ ID NO: 891) 215

TABLE 14 All hexamers that can be extracted from human D segments GIVGAT D1-26.1.1 (SEQ ID NO: 282) 1 VyWELL D1-26.2.1 (SEQ ID NO: 283) 2 YSGSYY D1-26.3.1 (SEQ ID NO: 284) 3 RILyyY D2-2.1.1 (SEQ ID NO: 300) 4 GYCSST D2-2.2.1 (SEQ ID NO: 301) 5 DIVVVP D2-2.3.1 (SEQ ID NO: 302) 6 ILyyYQ D2-2.1.2 (SEQ ID NO: 309) 7 YCSSTS D2-2.2.2 (SEQ ID NO: 310) 8 IVVVPA D2-2.3.2 (SEQ ID NO: 311) 9 LyyYQL D2-2.1.3 (SEQ ID NO: 318) 10 CSSTSC D2-2.2.3 (SEQ ID NO: 319) 11 VVVPAA D2-2.3.3 (SEQ ID NO: 320) 12 yyYQLL D2-2.1.4 (SEQ ID NO: 327) 13 SSTSCY D2-2.2.4 (SEQ ID NO: 328) 14 VVPAAI D2-2.3.4 (SEQ ID NO: 329) 15 yYQLLY D2-2.1.5 (SEQ ID NO: 336) 16 STSCYT D2-2.2.5 (SEQ ID NO: 337) 17 RILYyW D2-8.1.1 (SEQ ID NO: 351) 18 GYCTNG D2-8.2.1 (SEQ ID NO: 352) 19 DIVLMV D2-8.3.1 (SEQ ID NO: 353) 20 ILYyWC D2-8.1.2 (SEQ ID NO: 360) 21 YCTNGV D2-8.2.2 (SEQ ID NO: 361) 22 IVLMVY D2-8.3.2 (SEQ ID NO: 362) 23 LYyWCM D2-8.1.3 (SEQ ID NO: 369) 24 CTNGVC D2-8.2.3 (SEQ ID NO: 370) 25 VLMVYA D2-8.3.3 (SEQ ID NO: 371) 26 YyWCML D2-8.1.4 (SEQ ID NO: 378) 27 TNGVCY D2-8.2.4 (SEQ ID NO: 379) 28 LMVYAI D2-8.3.4 (SEQ ID NO: 380) 29 yWCMLY D2-8.1.5 (SEQ ID NO: 387) 30 NGVCYT D2-8.2.5 (SEQ ID NO: 388) 31 RILyWW D2-15.1.1 (SEQ ID NO: 398) 32 GYCSGG D2-15.2.1 (SEQ ID NO: 399) 33 DIVVVV D2-15.3.1 (SEQ ID NO: 400) 34 ILyWWy D2-15.1.2 (SEQ ID NO: 406) 35 YCSGGS D2-15.2.2 (SEQ ID NO: 407) 36 IVVVVA D2-15.3.2 (SEQ ID NO: 408) 37 LyWWyL D2-15.1.3 (SEQ ID NO: 415) 38 CSGGSC D2-15.2.3 (SEQ ID NO: 416) 39 VVVVAA D2-15.3.3 (SEQ ID NO: 417) 40 yWWyLL D2-15.1.4 (SEQ ID NO: 424) 41 SGGSCY D2-15.2.4 (SEQ ID NO: 425) 42 VVVAAT D2-15.3.4 (SEQ ID NO: 426) 43 WWyLLL D2-15.1.5 (SEQ ID NO: 433) 44 GGSCYS D2-15.2.5 (SEQ ID NO: 434) 45 SILWWw D2-21.1.1 (SEQ ID NO: 448) 46 AYCGGD D2-21.2.1 (SEQ ID NO: 449) 47 HIVVVT D2-21.3.1 (SEQ ID NO: 450) 48 ILWWwL D2-21.1.2 (SEQ ID NO: 456) 49 YCGGDC D2-21.2.2 (SEQ ID NO: 457) 50 IVVVTA D2-21.3.2 (SEQ ID NO: 458) 51 LWWwLL D2-21.1.3 (SEQ ID NO: 465) 52 CGGDCY D2-21.2.3 (SEQ ID NO: 466) 53 VVVTAI D2-21.3.3 (SEQ ID NO: 467) 54 WWwLLF D2-21.1.4 (SEQ ID NO: 474) 55 GGDCYS D2-21.2.4 (SEQ ID NO: 475) 56 VLRFLE D3-3.1.1 (SEQ ID NO: 489) 57 YYDFWS D3-3.2.1 (SEQ ID NO: 490) 58 ITIFGV D3-3.3.1 (SEQ ID NO: 491) 59 LRFLEW D3-3.1.2 (SEQ ID NO: 498) 60 YDFWSG D3-3.2.2 (SEQ ID NO: 499) 61 TIFGVV D3-3.3.2 (SEQ ID NO: 500) 62 RFLEWL D3-3.1.3 (SEQ ID NO: 507) 63 DFWSGY D3-3.2.3 (SEQ ID NO: 508) 64 IFGVVI D3-3.3.3 (SEQ ID NO: 509) 65 FLEWLL D3-3.1.4 (SEQ ID NO: 516) 66 FWSGYY D3-3.2.4 (SEQ ID NO: 517) 67 FGVVII D3-3.3.4 (SEQ ID NO: 518) 68 LEWLLY D3-3.1.5 (SEQ ID NO: 525) 69 WSGYYT D3-3.2.5 (SEQ ID NO: 526) 70 VLRYFD D3-9.1.1 (SEQ ID NO: 539) 71 YYDILT D3-9.2.1 (SEQ ID NO: 540) 72 ITIFyL D3-9.3.1 (SEQ ID NO: 541) 73 LRYFDW D3-9.1.2 (SEQ ID NO: 548) 74 YDILTG D3-9.2.2 (SEQ ID NO: 549) 75 TIFyLV D3-9.3.2 (SEQ ID NO: 550) 76 RYFDWL D3-9.1.3 (SEQ ID NO: 557) 77 DILTGY D3-9.2.3 (SEQ ID NO: 558) 78 IFyLVI D3-9.3.3 (SEQ ID NO: 559) 79 YFDWLL D3-9.1.4 (SEQ ID NO: 566) 80 ILTGYY D3-9.2.4 (SEQ ID NO: 567) 81 FyLVII D3-9.3.4 (SEQ ID NO: 568) 82 FDWLLy D3-9.1.5 (SEQ ID NO: 575) 83 LTGYYN D3-9.2.5 (SEQ ID NO: 576) 84 VLLWFG D3-10.1.1 (SEQ ID NO: 590) 85 YYYGSG D3-10.2.1 (SEQ ID NO: 591) 86 ITMVRG D3-10.3.1 (SEQ ID NO: 592) 87 LLWFGE D3-10.1.2 (SEQ ID NO: 599) 88 YYGSGS D3-10.2.2 (SEQ ID NO: 600) 89 TMVRGV D3-10.3.2 (SEQ ID NO: 601) 90 LWFGEL D3-10.1.3 (SEQ ID NO: 608) 91 YGSGSY D3-10.2.3 (SEQ ID NO: 609) 92 MVRGVI D3-10.3.3 (SEQ ID NO: 610) 93 WFGELL D3-10.1.4 (SEQ ID NO: 617) 94 GSGSYY D3-10.2.4 (SEQ ID NO: 618) 95 VRGVII D3-10.3.4 (SEQ ID NO: 619) 96 FGELLy D3-10.1.5 (SEQ ID NO: 624) 97 SGSYYN D3-10.2.5 (SEQ ID NO: 625) 98 VLwLRL D3-16.1.1 (SEQ ID NO: 638) 99 YYDYVW D3-16.2.1 (SEQ ID NO: 639) 100 IMITFG D3-16.3.1 (SEQ ID NO: 640) 101 LwLRLG D3-16.1.2 (SEQ ID NO: 647) 102 YDYVWG D3-16.2.2 (SEQ ID NO: 648) 103 MITFGG D3-16.3.2 (SEQ ID NO: 649) 104 wLRLGE D3-16.1.3 (SEQ ID NO: 656) 105 DYVWGS D3-16.2.3 (SEQ ID NO: 657) 106 ITFGGV D3-16.3.3 (SEQ ID NO: 658) 107 LRLGEL D3-16.1.4 (SEQ ID NO: 665) 108 YVWGSY D3-16.2.4 (SEQ ID NO: 666) 109 TFGGVI D3-16.3.4 (SEQ ID NO: 667) 110 RLGELS D3-16.1.5 (SEQ ID NO: 674) 111 VWGSYR D3-16.2.5 (SEQ ID NO: 675) 112 FGGVIV D3-16.3.5 (SEQ ID NO: 676) 113 LGELSL D3-16.1.6 (SEQ ID NO: 683) 114 WGSYRY D3-16.2.6 (SEQ ID NO: 684) 115 GGVIVI D3-16.3.6 (SEQ ID NO: 685) 116 GELSLY D3-16.1.7 (SEQ ID NO: 692) 117 GSYRYT D3-16.2.7 (SEQ ID NO: 693) 118 VLLwyy D3-22.1.1 (SEQ ID NO: 707) 119 YYYDSS D3-22.2.1 (SEQ ID NO: 708) 120 ITMIVV D3-22.3.1 (SEQ ID NO: 709) 121 LLwyyW D3-22.1.2 (SEQ ID NO: 716) 122 YYDSSG D3-22.2.2 (SEQ ID NO: 717) 123 TMIVVV D3-22.3.2 (SEQ ID NO: 718) 124 LwyyWL D3-22.1.3 (SEQ ID NO: 725) 125 YDSSGY D3-22.2.3 (SEQ ID NO: 726) 126 MIVVVI D3-22.3.3 (SEQ ID NO: 727) 127 wyyWLL D3-22.1.4 (SEQ ID NO: 733) 128 DSSGYY D3-22.2.4 (SEQ ID NO: 734) 129 IVVVIT D3-22.3.4 (SEQ ID NO: 735) 130 yyWLLL D3-22.1.5 (SEQ ID NO: 742) 131 SSGYYY D3-22.2.5 (SEQ ID NO: 743) 132 wLRWyL D4-23.1.1 (SEQ ID NO: 769) 133 DYGGNS D4-23.2.1 (SEQ ID NO: 770) 134 VDTAMV D5-5.1.1 (SEQ ID NO: 784) 135 WIQLWL D5-5.2.1 (SEQ ID NO: 785) 136 GYSYGY D5-5.3.1 (SEQ ID NO: 786) 137 VDIVAT D5-12.1.1 (SEQ ID NO: 802) 138 WIyWLR D5-12.2.1 (SEQ ID NO: 803) 139 GYSGYD D5-12.3.1 (SEQ ID NO: 804) 140 DIVATI D5-12.1.2 (SEQ ID NO: 811) 141 IyWLRL D5-12.2.2 (SEQ ID NO: 812) 142 YSGYDY D5-12.3.2 (SEQ ID NO: 813) 143 VEMATI D5-24.1.1 (SEQ ID NO: 829) 144 yRWLQL D5-24.2.1 (SEQ ID NO: 830) 145 RDGYNY D5-24.3.1 (SEQ ID NO: 831) 146 EYSSSS D6-6.1.1 (SEQ ID NO: 847) 147 GYSSSW D6-13.1.1 (SEQ ID NO: 859) 148 GIAAAG D6-13.2.1 (SEQ ID NO: 860) 149 VyQQLV D6-13.3.1 (SEQ ID NO: 861) 150 YSSSWY D6-13.1.2 (SEQ ID NO: 867) 151 GYSSGW D6-19.1.1 (SEQ ID NO: 878) 152 GIAVAG D6-19.2.1 (SEQ ID NO: 879) 153 VyQWLV D6-19.3.1 (SEQ ID NO: 880) 154 YSSGWY D6-19.1.2 (SEQ ID NO: 887) 155

Example 3: CDR3 of Length 6-20

Insertion of D segments into synthetic HC CDR3s can lead to greater stability and lower immunogenicity. Libraries are designed at the amino-acid level by joining a VH to an optional filler of some length which is joined to a D segment an optional second filler and a JH. For libraries of length six or eight, a full-length JH may follow VH and a short filler. Table 77 shows the frequency of D segments in a sampling of 1419 Abs selected from FAB-310 or FAB-410 for binding to one target or another. In the sample, 1099 Abs had no detectable D segment (i.e., less that 70% match). Where D segments are used, the D segments D1-1.3, D1-26.3, D2-2.2, D2-8.2, D2-15.2, D2-21.2, D3-16.2, D3-22.2, D3-3.2, D3-9.1, D3-9.2, D3-10.2, D3-16.2, D4-4.2, D4-4.3, D4-11.2, D4-4.2, D4-17.2, D4-23.2, D5-5.3, D5-12.3, D5-18.3, D6-6.1, D6-6.2, D6-13.1, D6-13.2, D6-19.1, D6-19.2, and D7-27.1 are preferred.

Once the parental amino-acid sequence has been designed, it can be diversified in several ways: error-prone PCR, wobbling, and dobbling. Table 14 shows a number of hexamers that can be derived from human D regions. In one embodiment, the hexamers that contain cysteine residues are exclused. In one embodiment, the fragments of D regions that contain stops are excluded. In one embodiment, any TAG codon found in the D region is replaced by a codon picked from the set comprising TCG, TTG, TGG, CAG, AAG, TAT, and GAG. In one embodiment, any TAA codon found in the D region is replaced by a codon picked form the set comprising TCA, TTA, CAA, AAA, TAT, and GAA. In one embodiment, any TGA of the D region is replaced by a codon picked from the set comprising TGG, TCA, TTA, AGA, and GGA.

Table 21 shows exemplary parental amino-acid sequences for CDR3s from 6 to 20 amino acids. These parental sequences can be combined with diversity in HC CDR1 and CDR2 to form a library. The utility is likely to improve if the CDR3 regions are diversified by, for example, wobbling, dobbling, or error-prone PCR of the CDR3s. In Table 21, sequence 6a comprises the end of VH from 3-23 fused to whole JH1. Sequence 6b contains the end of 3-23 joined to a Y joined to D4-17 (RF 2) joined to the FR4 region of JH1. Sequence 6c contains the end of 3-23 followed by D5-5 (RF 3) followed by the FR4 part of JH1. Sequence 6d contains the end of 3-23 joined to SY joined to the whole JH4. Table 21 shows the level of doping that would be appropriate for the wobbling of the CDR3; other levels could be used as well. Other D regions or fragments of D regions could be used. Other JH sequences could be used.

TABLE 21 Parental amino-acid sequences for HC CDR3s of 6-20 AAs. level of SEQ Length Parental sequence doping Comment ID NO:  6a yycakAEYFQHwgqgtlvtvss 70:10:10:10 JH1(whole) 226  6b yycakYDYGDYwgqgtlvtvss 70:10:10:10 Y::D4-17(2)::FR4 of JH1 227  6c yycakGYSYGYwgqgtlvtvss 70:10:10:10 D5-5(3)::FR4 of JH1 228  6d yycakSYYFDYwgqgtlvtvss 70:10:10:10 SY::JH4(whole) 229  8a yycakYYAEYFQHwgqgtlvtvss 73:9:9:9 YY:JH1(whole) 230  8b yycakYGYSSSWYwgqgtlvtvss 73:9:9:9 Y::D6-13(1)::FR4 of JH1 231  8c yycakYGDYYFDYwgqgtlvtvss 73:9:9:9 D4-17(2)[2-5]::JH4(whole) 232 10a yycakYYYDSSGYYYwgqgtlvtvss 73:9:9:9 D3-22(2)::Fr4 of JH1 233 10b yycakGYcSSTScYTwgqgtlvtvss 73:9:9:9 D2-2(2)::Fr4 of JH1 234 10c yycakYYSSAEYFQHwgqgtlvtvss 73:9:9:9 YYSS::JH1(whole) 235 (SEQ ID NO: 942) 10d yycakGYSYGYYFDYwgqgtlvtvss 73:9:9:9 D5-5(3)::JH4(whole) 236 12a yycakYYYDSSGYYYQHwgqgtlvtvss 85:5:5:5 D3-22(2)::QH::Fr4 of JH1 237 12b yycakGYcSSTScYTQHwgqgtlvtvss 85:5:5:5 D2-2(2)::QH::Fr4 of JH1 238 12c yycakYDGSYSAEYFQHwgqgtlvtvss 85:5:5:5 YDGSYS::JH1(whole) 239 (SEQ ID NO: 943) 12d yycakYYDYVWGSYRYTwgqgtlvtvss 85:5:5:5 D3-16(2)::Fr of JH1 240 12e yycakGYSYGYYWYFDLwgrgtlvtvss 85:5:5:5 D5-5(3)::JH2(whole) 241 14a yycakYYYDSSGYYYYFQHwgqgtlvtvss 73:9:9:9 D3-22(2)::YFQH::Fr of JH1 242 (SEQ ID NO: 944) 14b yycakGYcSSTScYTYFQHwgqgtlvtvss 73:9:9:9 D2-2(2)::YFQH::Fr of JH1 243 (SEQ ID NO: 944) 14c yycakSYGYcSSTScYTQHwgqgtlvtvss 73:9:9:9 SY::D2-2(2)::QH::Fr of JH1 244 14d yycakSYRYSGYSAEYFQHwgqgtlvtvss 73:9:9:9 SYRYSGYS::JH1(whole) 245 (SEQ ID NO: 945) 14e yycakAYcGGDcYSNWFDPwgqgtlvtvss 73:9:9:9 D2-21(2)::JH5(whole) 246 15a yycakSDGYYYDSSGYYYDYwgqgtlvtvss 73:9:9:9 SD::D3-22.2::JH4(101ff) 930 15b yycakGSGYCSGGSCYSFDYwgqgtlvtvss 73:9:9:9 GS::D2-15.2::JH4(100ff) 931 15c yycakGGRGYSSGWYRAFDIwgqgtmvtvss 73:9:9:9 GGR::D6-19.1::R::JH3(all) 932 16a yycakYYYDSSGYYYAEYFQHwgqgtlvtvss 73:9:9:9 D3-22(2)::JH1(whole) 247 16b yycakGYcSSTScYTAEYFQHwgqgtlvtvss 73:9:9:9 D2-2(2)::JH1(whole) 248 16c yycakSYDSYRSYGSAEYFQHwgqgtlvtvss 73:9:9:9 SYDSYRSYGS::JH1(whole) 249 (SEQ ID NO: 946) 16d yycakSYSYGYcSSTScYTQHwgqgtlvtvss 73:9:9:9 SYSY::D2-2(2)::QH::Fr JH1 250 (SEQ ID NO: 947) 17a yycakSRPGYSSSWYYYYGMDVwgqgttvtvss 73:9:9:9 SRP::6-13.1::JH6(-1Y) 933 18a yycakGYCSGGSCYSYYYYGMDVwgqgttvtvss 73:9:9:9 2-15.2::JH6(-1Y) 221 18b yycakDGYCSGGSCYSYYYGMDVwgqgttvtvss 73:9:9:9 D::2-15.2::JH6(-2Ys) 222 19a yycakDGYYYDSSGYYYRGYYFDYwgqgtlvtvss 73:9:9:9 D::D3-22.2::RGY::JH4(all) 223 20a yycakYSSYYYYDSSGYYYAEYFQHwgqgtlvtvss 73:9:9:9 YSSY::D3-22(2)::JH1(whole) 251 (SEQ ID NO: 948) 20b yycakSYYSGYcSSTScYTAEYFQHwgqgtlvtvss 73:9:9:9 SYYS::D2-2(2)::JH1(whole) 252 (SEQ ID NO: 949) 20c yycakSGYcSSTScYTYYSAEYFQHwgqgtlvtvss 73:9:9:9 S::D2-2(2)::YYS::JH1(whole) 253 20d yycakYYYYDYVWGSYRYTSNWFDPwgqgtlvtvss 73:9:9:9 Y::D3-16(2)::S::JH5(whole) 254 20e yycakYYYYDYVWGSYRYTSSYFDYwgqgtlvtvss 73:9:9:9 Y::D3-16(2)::SS::JH4(whole) 255

TABLE 22 HC display cassette The amino-acid sequence shown in Table 22 is SEQ ID NO: 892. The DNA sequence shown in Table 22 is SEQ ID NO: 893. !  Signal for VH-CH1-IIIstump !   1   2   3   4   5   6   7   8   9  10  11  12  13  14  15 !  M   K   Y   L   L   P   T   A   A   A   G   L   L   L   L  946 atg aaa tac cta ttg cct acg gca gcc gct gga ttg tta tta ctc ! !  16  17  18  19  20  21  22 !  A   A   Q   P   A   M   A  991 gcG GCC cag ccG GCC atg gcc !   SfiI............. !           NgoMI...(1/2) !                  NcoI.... ! ! VH !                              FR1(DP47/V3-23)--------------- !                               1   2   3   4   5   6   7   8 !                               E   V   Q   L   L   E   S   G 1012                              gaa|gtt|CAA|TTG|tta|gag|tct|ggt| !                                     | MfeI  | ! ! --------------FR1-------------------------------------------- !   9  10  11  12  13  14  15  16  17  18  19  20  21  22  23 !   G   G   L   V   Q   P   G   G   S   L   R   L   S   C   A 1036 |ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta|cgt|ctt|tct|tgc|gct| ! ! ----FR1-------------------->|...CDR1............|---FR2------ !  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38 !   A   S   G   F   T   F   S   S   Y   A   M   S   W   V   R 1081 |gct|TCC|GGA|ttc|act|ttc|tct|tCG|TAC|Gct|atg|tct|tgg|gtt|cgC| !     | BspEI |                 | BsiWI|                     |BstXI. ! ! --------FR2-------------------------------->|...CDR2......... !  39  40  41  42  43  44  45  46  47  48  49  50  51  52  52a !   Q   A   P   G   K   G   L   E   W   V   S   A   I   S   G 1126 |CAa|gct|ccT|GGt|aaa|ggt|ttg|gag|tgg|gtt|tct|gct|atc|tct|ggt| !      ...BstXI          | ! ! ....CDR2............................................|---FR3--- !  53  54  55  56  57  58  59  60  61  62  63  64  65  66  67 !   S   G   G   S   T   Y   Y   A   D   S   V   K   G   R   F 1171 |tct|ggt|ggc|agt|act|tac|tat|gct|gac|tcc|gtt|aaa|ggt|cgc|ttc| ! ! ! --------FR3-------------------------------------------------- !  68  69  70  71  72  73  74  75  76  77  78  79  80  81  82 !   T   I   S   R   D   N   S   K   N   T   L   Y   L   Q   M 1216 |act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg| !         | XbaI  | ! !  ---FR3---------------------------------------------------->| !  82a 82b 82c 83  84  85  86  87  88  89  90  91  92  93  94 !   N   S   L   R   A   E   D   T   A   V   Y   Y   C   A   K 1261 |aac|agC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgc|gct|aaa| !        |AflII |               | PstI |(2/2) ! ! .......CDR3.................................|----FR4-------- !  95  96  97  98 98a 98b 98c  99  100 101 102 103 104 105 106 !   D   Y   E   G   T   G   Y   A   F   D   I   W   G   Q   G 1306 |gac|tat|gaa|ggt|act|ggt|tat|gct|ttc|gaC|ATA|TGg|ggt|caa|ggt| !                                        | NdeI | ! ! --------------FR4---------->| !  107 108 109 110 111 112 113 !   T   M   V   T   V   S   S 1351 |act|atG|GTC|ACC|gtc|tct|agt !        | BstEII |  c tcg ag = XhoI. ! ! CH1 !  A   S   T   K   G   P   S   V   F   P   L   A   P   S   S 1372 gcc tcc acc aag ggc cca tcg gtc ttc ccG CTA GCa ccc tcc tcc !                                       NheI.... ! ! 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 !  K   S   T   S   G   G   T   A   A   L   G   C   L   V   K 1417 aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag ! ! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 !  D   Y   F   P   E   P   V   T   V   S   W   N   S   G   A 1462 gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ! ! 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 !  L   T   S   G   V   H   T   F   P   A   V   L   Q   S   S 1507 ctg acc agc ggc gtc cac acc ttc ccg gct gtc cta cag tcc tca ! ! 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 !  G   L   Y   S   L   S   S   V   V   T   V   P   S   S   S 1552 gga ctc tac tcc ctc agc agc gta gtg acc gtg ccc tCC Agc agc !                                                  BstXI........ ! ! 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 !  L   G   T   Q   T   Y   I   C   N   V   N   H   K   P   S 1597 tTG Ggc acc cag acc tac atc tgc aac gtg aat cac aag ccc agc !  BstXI........ ! ! 226 227 228 229 230 231 232 233 234 235 236 237 238 !  N   T   K   V   D   K   K   V   E   P   K   S   C 1642 aac acc aag gtg gac aaG AAA GTT GAG CCC AAA TCT TGT ! ! 139 140 141  His tag..............   cMyc tag...................... !  A   A   A   H   H   H   H   H   H   G   A   A   E   Q   K   L   I 1681 GCG GCC GCa cat cat cat cac cat cac ggg gcc gca gaa caa aaa ctc atc ! NotI...... !  EagI.... ! !   .................................. !  S   E   E   D   L   N   G   A   A   E   A   S   S   A   S   N   A   S 1732 tca gaa gag gat ctg aat ggg GCC gca gaG GCt agt tct gct agt aAC GCG Tct !                            BglI..........(3/4)              MluI.... ! !     Domain 3 (IIIstump)----------------------------------------------------- !  S   G   D   F   D   Y   E   K   M   A   N   A   N   K   G   A 1786 tcc ggt gat ttt gat tat gaa aag atg gca aac gct aat aag ggg gct ! !  M   T   E   N   A   D   E   N   A   L   Q   S   D   A   K   G 1834 atg acc gaa aat gcc gat gaa aac gcg cta cag tct gac gct aaa ggc ! !  K   L   D   S   V   A   T   D   Y   G   A   A   I   D   G   F 1882 aaa ctt gat tct gtc gct act gat tac ggt gct gct atc gat ggt ttc ! !  I   G   D   V   S   G   L   A   N   G   N   G   A   T   G   D 1930 att ggt gac gtt tcc ggc ctt gct aat ggt aat ggt gct act ggt gat ! !  F   A   G   S   N   S   Q   M   A   Q   V   G   D   G   D   N 1978 ttt gct ggc tct aat tcc caa atg gct caa gtc ggt gac ggt gat aat ! !  S   P   L   M   N   N   F   R   Q   Y   L   P   S   L   P   Q 2026 tca cct tta atg aat aat ttc cgt caa tat tta cct tcc ctc cct caa ! !  S   V   E   C   R   P   F   V   F   G   A   G   K   P   Y   E 2074 tcg gtt gaa tgt cgc cct ttt gtc ttt ggc gct ggt aaa cca tat gaa ! !  F   S   I   D   C   D   K   I   N   L   F   R 2122 ttt tct att gat tgt gac aaa ata aac tta ttc cgt !                                             End Domain 3 ! !  G   V   F   A   F   L   L   Y   V   A   T   F   M   Y   V  F140 2158 ggt gtc ttt gcg ttt ctt tta tat gtt gcc acc ttt atg tat gta ttt ! start transmembrane segment ! !  S   T   F   A   N   I   L 2206 tct acg ttt gct aac ata ctg ! !  R   N   K   E   S   (SEQ ID NO: 892) 2227 cgt aat aag gag tct TAA    tga aAC GCG Tga tga GAATTC (SEQ ID NO: 893) ! Intracellular anchor.           MluI....       EcoRI.

TABLE 25 The DNA sequence of DY3F85LC containing a sample germline  O12 kappa light chain. The antibody sequences shown are of the form of actual antibody, but have not been identified as binding to a particular antigen. On each line, everything after an exclamation point (!) is commentary. The DNA of DY3F85LC is SEQ ID NO: 950 !-----------------------------------------------------------------------------      1 AATGCTACTA CTATTAGTAG AATTGATGCC ACCTTTTCAG CTCGCGCCCC AAATGAAAAT     61 ATAGCTAAAC AGGTTATTGA CCATTTGCGA AATGTATCTA ATGGTCAAAC TAAATCTACT    121 CGTTCGCAGA ATTGGGAATC AACTGTTATA TGGAATGAAA CTTCCAGACA CCGTACTTTA    181 GTTGCATATT TAAAACATGT TGAGCTACAG CATTATATTC AGCAATTAAG CTCTAAGCCA    241 TCCGCAAAAA TGACCTCTTA TCAAAAGGAG CAATTAAAGG TACTCTCTAA TCCTGACCTG    301 TTGGAGTTTG CTTCCGGTCT GGTTCGCTTT GAAGCTCGAA TTAAAACGCG ATATTTGAAG    361 TCTTTCGGGC TTCCTCTTAA TCTTTTTGAT GCAATCCGCT TTGCTTCTGA CTATAATAGT    421 CAGGGTAAAG ACCTGATTTT TGATTTATGG TCATTCTCGT TTTCTGAACT GTTTAAAGCA    481 TTTGAGGGGG ATTCAATGAA TATTTATGAC GATTCCGCAG TATTGGACGC TATCCAGTCT    541 AAACATTTTA CTATTACCCC CTCTGGCAAA ACTTCTTTTG CAAAAGCCTC TCGCTATTTT    601 GGTTTTTATC GTCGTCTGGT AAACGAGGGT TATGATAGTG TTGCTCTTAC TATGCCTCGT    661 AATTCCTTTT GGCGTTATGT ATCTGCATTA GTTGAATGTG GTATTCCTAA ATCTCAACTG    721 ATGAATCTTT CTACCTGTAA TAATGTTGTT CCGTTAGTTC GTTTTATTAA CGTAGATTTT    781 TCTTCCCAAC GTCCTGACTG GTATAATGAG CCAGTTCTTA AAATCGCATA AGGTAATTCA    841 CAATGATTAA AGTTGAAATT AAACCATCTC AAGCCCAATT TACTACTCGT TCTGGTGTTT    901 CTCGTCAGGG CAAGCCTTAT TCACTGAATG AGCAGCTTTG TTACGTTGAT TTGGGTAATG    961 AATATCCGGT TCTTGTCAAG ATTACTCTTG ATGAAGGTCA GCCAGCCTAT GCGCCTGGTC   1021 TGTACACCGT TCATCTGTCC TCTTTCAAAG TTGGTCAGTT CGGTTCCCTT ATGATTGACC   1081 GTCTGCGCCT CGTTCCGGCT AAGTAACATG GAGCAGGTCG CGGATTTCGA CACAATTTAT   1141 CAGGCGATGA TACAAATCTC CGTTGTACTT TGTTTCGCGC TTGGTATAAT CGCTGGGGGT   1201 CAAAGATGAG TGTTTTAGTG TATTCTTTTG CCTCTTTCGT TTTAGGTTGG TGCCTTCGTA   1261 GTGGCATTAC GTATTTTACC CGTTTAATGG AAACTTCCTC ATGAAAAAGT CTTTAGTCCT   1321 CAAAGCCTCT GTAGCCGTTG CTACCCTCGT TCCGATGCTG TCTTTCGCTG CTGAGGGTGA   1381 CGATCCCGCA AAAGCGGCCT TTAACTCCCT GCAAGCCTCA GCGACCGAAT ATATCGGTTA   1441 TGCGTGGGCG ATGGTTGTTG TCATTGTCGG CGCAACTATC GGTATCAAGC TGTTTAAGAA   1501 ATTCACCTCG AAAGCAAGCT GATAAACCGA TACAATTAAA GGCTCCTTTT GGAGCCTTTT   1561 TTTTGGAGAT TTTCAACGTG AAAAAATTAT TATTCGCAAT TCCTTTAGTT GTTCCTTTCT   1621 ATTCTCACTC CGCTGAAACT GTTGAAAGTT GTTTAGCAAA ATCCCATACA GAAAATTCAT   1681 TTACTAACGT CTGGAAAGAC GACAAAACTT TAGATCGTTA CGCTAACTAT GAGGGCTGTC   1741 TGTGGAATGC TACAGGCGTT GTAGTTTGTA CTGGTGACGA AACTCAGTGT TACGGTACAT   1801 GGGTTCCTAT TGGGCTTGCT ATCCCTGAAA ATGAGGGTGG TGGCTCTGAG GGTGGCGGTT   1861 CTGAGGGTGG CGGTTCTGAG GGTGGCGGTA CTAAACCTCC TGAGTACGGT GATACACCTA   1921 TTCCGGGCTA TACTTATATC AACCCTCTCG ACGGCACTTA TCCGCCTGGT ACTGAGCAAA   1981 ACCCCGCTAA TCCTAATCCT TCTCTTGAGG AGTCTCAGCC TCTTAATACT TTCATGTTTC   2041 AGAATAATAG GTTCCGAAAT AGGCAGGGGG CATTAACTGT TTATACGGGC ACTGTTACTC   2101 AAGGCACTGA CCCCGTTAAA ACTTATTACC AGTACACTCC TGTATCATCA AAAGCCATGT   2161 ATGACGCTTA CTGGAACGGT AAATTCAGAG ACTGCGCTTT CCATTCTGGC TTTAATGAGG   2221 ATTTATTTGT TTGTGAATAT CAAGGCCAAT CGTCTGACCT GCCTCAACCT CCTGTCAATG   2281 CTGGCGGCGG CTCTGGTGGT GGTTCTGGTG GCGGCTCTGA GGGTGGTGGC TCTGAGGGTG   2341 GCGGTTCTGA GGGTGGCGGC TCTGAGGGAG GCGGTTCCGG TGGTGGCTCT GGTTCCGGTG   2401 ATTTTGATTA TGAAAAGATG GCAAACGCTA ATAAGGGGGC TATGACCGAA AATGCCGATG   2461 AAAACGCGCT ACAGTCTGAC GCTAAAGGCA AACTTGATTC TGTCGCTACT GATTACGGTG   2521 CTGCTATCGA TGGTTTCATT GGTGACGTTT CCGGCCTTGC TAATGGTAAT GGTGCTACTG   2581 GTGATTTTGC TGGCTCTAAT TCCCAAATGG CTCAAGTCGG TGACGGTGAT AATTCACCTT   2641 TAATGAATAA TTTCCGTCAA TATTTACCTT CCCTCCCTCA ATCGGTTGAA TGTCGCCCTT   2701 TTGTCTTTGG CGCTGGTAAA CCATATGAAT TTTCTATTGA TTGTGACAAA ATAAACTTAT   2761 TCCGTGGTGT CTTTGCGTTT CTTTTATATG TTGCCACCTT TATGTATGTA TTTTCTACGT   2821 TTGCTAACAT ACTGCGTAAT AAGGAGTCTT AATCATGCCA GTTCTTTTGG GTATTCCGTT   2881 ATTATTGCGT TTCCTCGGTT TCCTTCTGGT AACTTTGTTC GGCTATCTGC TTACTTTTCT   2941 TAAAAAGGGC TTCGGTAAGA TAGCTATTGC TATTTCATTG TTTCTTGCTC TTATTATTGG   3001 GCTTAACTCA ATTCTTGTGG GTTATCTCTC TGATATTAGC GCTCAATTAC CCTCTGACTT   3061 TGTTCAGGGT GTTCAGTTAA TTCTCCCGTC TAATGCGCTT CCCTGTTTTT ATGTTATTCT   3121 CTCTGTAAAG GCTGCTATTT TCATTTTTGA CGTTAAACAA AAAATCGTTT CTTATTTGGA   3181 TTGGGATAAA TAATATGGCT GTTTATTTTG TAACTGGCAA ATTAGGCTCT GGAAAGACGC   3241 TCGTTAGCGT TGGTAAGATT CAGGATAAAA TTGTAGCTGG GTGCAAAATA GCAACTAATC   3301 TTGATTTAAG GCTTCAAAAC CTCCCGCAAG TCGGGAGGTT CGCTAAAACG CCTCGCGTTC   3361 TTAGAATACC GGATAAGCCT TCTATATCTG ATTTGCTTGC TATTGGGCGC GGTAATGATT   3421 CCTACGATGA AAATAAAAAC GGCTTGCTTG TTCTCGATGA GTGCGGTACT TGGTTTAATA   3481 CCCGTTCTTG GAATGATAAG GAAAGACAGC CGATTATTGA TTGGTTTCTA CATGCTCGTA   3541 AATTAGGATG GGATATTATT TTTCTTGTTC AGGACTTATC TATTGTTGAT AAACAGGCGC   3601 GTTCTGCATT AGCTGAACAT GTTGTTTATT GTCGTCGTCT GGACAGAATT ACTTTACCTT   3661 TTGTCGGTAC TTTATATTCT CTTATTACTG GCTCGAAAAT GCCTCTGCCT AAATTACATG   3721 TTGGCGTTGT TAAATATGGC GATTCTCAAT TAAGCCCTAC TGTTGAGCGT TGGCTTTATA   3781 CTGGTAAGAA TTTGTATAAC GCATATGATA CTAAACAGGC TTTTTCTAGT AATTATGATT   3841 CCGGTGTTTA TTCTTATTTA ACGCCTTATT TATCACACGG TCGGTATTTC AAACCATTAA   3901 ATTTAGGTCA GAAGATGAAA TTAACTAAAA TATATTTGAA AAAGTTTTCT CGCGTTCTTT   3961 GTCTTGCGAT TGGATTTGCA TCAGCATTTA CATATAGTTA TATAACCCAA CCTAAGCCGG   4021 AGGTTAAAAA GGTAGTCTCT CAGACCTATG ATTTTGATAA ATTCACTATT GACTCTTCTC   4081 AGCGTCTTAA TCTAAGCTAT CGCTATGTTT TCAAGGATTC TAAGGGAAAA TTAATTAATA   4141 GCGACGATTT ACAGAAGCAA GGTTATTCAC TCACATATAT TGATTTATGT ACTGTTTCCA   4201 TTAAAAAAGG TAATTCAAAT GAAATTGTTA AATGTAATTA ATTTTGTTTT CTTGATGTTT   4261 GTTTCATCAT CTTCTTTTGC TCAGGTAATT GAAATGAATA ATTCGCCTCT GCGCGATTTT   4321 GTAACTTGGT ATTCAAAGCA ATCAGGCGAA TCCGTTATTG TTTCTCCCGA TGTAAAAGGT   4381 ACTGTTACTG TATATTCATC TGACGTTAAA CCTGAAAATC TACGCAATTT CTTTATTTCT   4441 GTTTTACGTG CAAATAATTT TGATATGGTA GGTTCTAACC CTTCCATAAT TCAGAAGTAT   4501 AATCCAAACA ATCAGGATTA TATTGATGAA TTGCCATCAT CTGATAATCA GGAATATGAT   4561 GATAATTCCG CTCCTTCTGG TGGTTTCTTT GTTCCGCAAA ATGATAATGT TACTCAAACT   4621 TTTAAAATTA ATAACGTTCG GGCAAAGGAT TTAATACGAG TTGTCGAATT GTTTGTAAAG   4681 TCTAATACTT CTAAATCCTC AAATGTATTA TCTATTGACG GCTCTAATCT ATTAGTTGTT   4741 AGTGCTCCTA AAGATATTTT AGATAACCTT CCTCAATTCC TTTCAACTGT TGATTTGCCA   4801 ACTGACCAGA TATTGATTGA GGGTTTGATA TTTGAGGTTC AGCAAGGTGA TGCTTTAGAT   4861 TTTTCATTTG CTGCTGGCTC TCAGCGTGGC ACTGTTGCAG GCGGTGTTAA TACTGACCGC   4921 CTCACCTCTG TTTTATCTTC TGCTGGTGGT TCGTTCGGTA TTTTTAATGG CGATGTTTTA   4981 GGGCTATCAG TTCGCGCATT AAAGACTAAT AGCCATTCAA AAATATTGTC TGTGCCACGT   5041 ATTCTTACGC TTTCAGGTCA GAAGGGTTCT ATCTCTGTTG GCCAGAATGT CCCTTTTATT   5101 ACTGGTCGTG TGACTGGTGA ATCTGCCAAT GTAAATAATC CATTTCAGAC GATTGAGCGT   5161 CAAAATGTAG GTATTTCCAT GAGCGTTTTT CCTGTTGCAA TGGCTGGCGG TAATATTGTT   5221 CTGGATATTA CCAGCAAGGC CGATAGTTTG AGTTCTTCTA CTCAGGCAAG TGATGTTATT   5281 ACTAATCAAA GAAGTATTGC TACAACGGTT AATTTGCGTG ATGGACAGAC TCTTTTACTC   5341 GGTGGCCTCA CTGATTATAA AAACACTTCT CAGGATTCTG GCGTACCGTT CCTGTCTAAA   5401 ATCCCTTTAA TCGGCCTCCT GTTTAGCTCC CGCTCTGATT CTAACGAGGA AAGCACGTTA   5461 TACGTGCTCG TCAAAGCAAC CATAGTACGC GCCCTGTAGC GGCGCATTAA GCGCGGCGGG   5521 TGTGGTGGTT ACGCGCAGCG TGACCGCTAC ACTTGCCAGC GCCCTAGCGC CCGCTCCTTT   5581 CGCTTTCTTC CCTTCCTTTC TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG   5641 GGGGCTCCCT TTAGGGTTCC GATTTAGTGC TTTACGGCAC CTCGACCCCA AAAAACTTGA   5701 TTTGGGTGAT GGTTCACGTA GTGGGCCATC GCCCTGATAG ACGGTTTTTC GCCCTTTGAC   5761 GTTGGAGTCC ACGTTCTTTA ATAGTGGACT CTTGTTCCAA ACTGGAACAA CACTCAACCC   5821 TATCTCGGGC TATTCTTTTG ATTTATAAGG GATTTTGCCG ATTTCGGAAC CACCATCAAA   5881 CAGGATTTTC GCCTGCTGGG GCAAACCAGC GTGGACCGCT TGCTGCAACT CTCTCAGGGC   5941 CAGGCGGTGA AGGGCAATCA GCTGTTGCCC GTCTCACTGG TGAAAAGAAA AACCACCCTG   6001 GATCCAAGCT TGCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT ATTTGTTTAT   6061 TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA TAAATGCTTC   6121 AATAATATTG AAAAAGGAAG AGTATGAGTA TTCAACATTT CCGTGTCGCC CTTATTCCCT   6181 TTTTTGCGGC ATTTTGCCTT CCTGTTTTTG CTCACCCAGA AACGCTGGTG AAAGTAAAAG   6241 ATGCTGAAGA TCAGTTGGGC GCACTAGTGG GTTACATCGA ACTGGATCTC AACAGCGGTA   6301 AGATCCTTGA GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT GATGAGCACT TTTAAAGTTC   6361 TGCTATGTGG CGCGGTATTA TCCCGTATTG ACGCCGGGCA AGAGCAACTC GGTCGCCGCA   6421 TACACTATTC TCAGAATGAC TTGGTTGAGT ACTCACCAGT CACAGAAAAG CATCTTACGG   6481 ATGGCATGAC AGTAAGAGAA TTATGCAGTG CTGCCATAAC CATGAGTGAT AACACTGCGG   6541 CCAACTTACT TCTGACAACG ATCGGAGGAC CGAAGGAGCT AACCGCTTTT TTGCACAACA   6601 TGGGGGATCA TGTAACTCGC CTTGATCGTT GGGAACCGGA GCTGAATGAA GCCATACCAA   6661 ACGACGAGCG TGACACCACG ATGCCTGTAG CAATGGCAAC AACGTTGCGC AAACTATTAA   6721 CTGGCGAACT ACTTACTCTA GCTTCCCGGC AACAATTAAT AGACTGGATG GAGGCGGATA   6781 AAGTTGCAGG ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTGGTTTATT GCTGATAAAT   6841 CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA GATGGTAAGC   6901 CCTCCCGTAT CGTAGTTATC TACACGACGG GGAGTCAGGC AACTATGGAT GAACGAAATA   6961 GACAGATCGC TGAGATAGGT GCCTCACTGA TTAAGCATTG GTAACTGTCA GACCAAGTTT   7021 ACTCATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG ATCTAGGTGA   7081 AGATCCTTTT TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG TTCCACTGTA   7141 CGTAAGACCC CCAAGCTTGT CGACTGAATG GCGAATGGCG CTTTGCCTGG TTTCCGGCAC   7201 CAGAAGCGGT GCCGGAAAGC TGGCTGGAGT GCGATCTTCC TGACGCTCGA GCGCAACGCA !                                                  XhoI...   7261 ATTAATGTGA GTTAGCTCAC TCATTAGGCA CCCCAGGCTT TACACTTTAT GCTTCCGGCT   7321 CGTATGTTGT GTGGAATTGT GAGCGGATAA CAATTTCACA CAGGAAACAG CTATGACCAT   7381 GATTACGCCA AGCTTTGGAG CCTTTTTTTT GGAGATTTTC AAC

TABLE 30 DNA sequence of DY3FHC87 (SEQ ID NO: 894)    1 aatgctacta ctattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat   61 atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact  121 cgttcgcaga attgggaatc aactgttata tggaatgaaa cttccagaca ccgtacttta  181 gttgcatatt taaaacatgt tgagctacag cattatattc agcaattaag ctctaagcca  241 tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactctctaa tcctgacctg  301 ttggagtttg cttccggtct ggttcgcttt gaagctcgaa ttaaaacgcg atatttgaag  361 tctttcgggc ttcctcttaa tctttttgat gcaatccgct ttgcttctga ctataatagt  421 cagggtaaag acctgatttt tgatttatgg tcattctcgt tttctgaact gtttaaagca  481 tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct  541 aaacatttta ctattacccc ctctggcaaa acttcttttg caaaagcctc tcgctatttt  601 ggtttttatc gtcgtctggt aaacgagggt tatgatagtg ttgctcttac tatgcctcgt  661 aattcctttt ggcgttatgt atctgcatta gttgaatgtg gtattcctaa atctcaactg  721 atgaatcttt ctacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt  781 tcttcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca  841 caatgattaa agttgaaatt aaaccatctc aagcccaatt tactactcgt tctggtgttt  901 ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg  961 aatatccggt tcttgtcaag attactcttg atgaaggtca gccagcctat gcgcctggtc 1021 tgtacaccgt tcatctgtcc tctttcaaag ttggtcagtt cggttccctt atgattgacc 1081 gtctgcgcct cgttccggct aagtaacatg gagcaggtcg cggatttcga cacaatttat 1141 caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt 1201 caaagatgag tgttttagtg tattcttttg cctctttcgt tttaggttgg tgccttcgta 1261 gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1321 caaagcctct gtagccgttg ctaccctcgt tccgatgctg tctttcgctg ctgagggtga 1381 cgatcccgca aaagcggcct ttaactccct gcaagcctca gcgaccgaat atatcggtta 1441 tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa 1501 attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1561 tttttggaga ttttcaacgt gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc 1621 tattctcact ccgctgaaac tgttgaaagt tgtttagcaa aatcccatac agaaaattca 1681 tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta tgagggctgt 1741 ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg aaactcagtg ttacggtaca 1801 tgggttccta ttgggcttgc tatccctgaa aatgagggtg gtggctctga gggtggcggt 1861 tctgagggtg gcggttctga gggtggcggt actaaacctc ctgagtacgg tgatacacct 1921 attccgggct atacttatat caaccctctc gacggcactt atccgcctgg tactgagcaa 1981 aaccccgcta atcctaatcc ttctcttgag gagtctcagc ctcttaatac tttcatgttt 2041 cagaataata ggttccgaaa taggcagggg gcattaactg tttatacggg cactgttact 2101 caaggcactg accccgttaa aacttattac cagtacactc ctgtatcatc aaaagccatg 2161 tatgacgctt actggaacgg taaattcaga gactgcgctt tccattctgg ctttaatgag 2221 gatttatttg tttgtgaata tcaaggccaa tcgtctgacc tgcctcaacc tcctgtcaat 2281 gctggcggcg gctctggtgg tggttctggt ggcggctctg agggtggtgg ctctgagggt 2341 ggcggttctg agggtggcgg ctctgaggga ggcggttccg gtggtggctc tggttccggt 2401 gattttgatt atgaaaagat ggcaaacgct aataaggggg ctatgaccga aaatgccgat 2461 gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt 2521 gctgctatcg atggtttcat tggtgacgtt tccggccttg ctaatggtaa tggtgctact 2581 ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga taattcacct 2641 ttaatgaata atttccgtca atatttacct tccctccctc aatcggttga atgtcgccct 2701 tttgtctttg gcgctggtaa accatatgaa ttttctattg attgtgacaa aataaactta 2761 ttccgtggtg tctttgcgtt tcttttatat gttgccacct ttatgtatgt attttctacg 2821 tttgctaaca tactgcgtaa taaggagtct taatcatgcc agttcttttg ggtattccgt 2881 tattattgcg tttcctcggt ttccttctgg taactttgtt cggctatctg cttacttttc 2941 ttaaaaaggg cttcggtaag atagctattg ctatttcatt gtttcttgct cttattattg 3001 ggcttaactc aattcttgtg ggttatctct ctgatattag cgctcaatta ccctctgact 3061 ttgttcaggg tgttcagtta attctcccgt ctaatgcgct tccctgtttt tatgttattc 3121 tctctgtaaa ggctgctatt ttcatttttg acgttaaaca aaaaatcgtt tcttatttgg 3181 attgggataa ataatatggc tgtttatttt gtaactggca aattaggctc tggaaagacg 3241 ctcgttagcg ttggtaagat tcaggataaa attgtagctg ggtgcaaaat agcaactaat 3301 cttgatttaa ggcttcaaaa cctcccgcaa gtcgggaggt tcgctaaaac gcctcgcgtt 3361 cttagaatac cggataagcc ttctatatct gatttgcttg ctattgggcg cggtaatgat 3421 tcctacgatg aaaataaaaa cggcttgctt gttctcgatg agtgcggtac ttggtttaat 3481 acccgttctt ggaatgataa ggaaagacag ccgattattg attggtttct acatgctcgt 3541 aaattaggat gggatattat ttttcttgtt caggacttat ctattgttga taaacaggcg 3601 cgttctgcat tagctgaaca tgttgtttat tgtcgtcgtc tggacagaat tactttacct 3661 tttgtcggta ctttatattc tcttattact ggctcgaaaa tgcctctgcc taaattacat 3721 gttggcgttg ttaaatatgg cgattctcaa ttaagcccta ctgttgagcg ttggctttat 3781 actggtaaga atttgtataa cgcatatgat actaaacagg ctttttctag taattatgat 3841 tccggtgttt attcttattt aacgccttat ttatcacacg gtcggtattt caaaccatta 3901 aatttaggtc agaagatgaa attaactaaa atatatttga aaaagttttc tcgcgttctt 3961 tgtcttgcga ttggatttgc atcagcattt acatatagtt atataaccca acctaagccg 4021 gaggttaaaa aggtagtctc tcagacctat gattttgata aattcactat tgactcttct 4081 cagcgtctta atctaagcta tcgctatgtt ttcaaggatt ctaagggaaa attaattaat 4141 agcgacgatt tacagaagca aggttattca ctcacatata ttgatttatg tactgtttcc 4201 attaaaaaag gtaattcaaa tgaaattgtt aaatgtaatt aattttgttt tcttgatgtt 4261 tgtttcatca tcttcttttg ctcaggtaat tgaaatgaat aattcgcctc tgcgcgattt 4321 tgtaacttgg tattcaaagc aatcaggcga atccgttatt gtttctcccg atgtaaaagg 4381 tactgttact gtatattcat ctgacgttaa acctgaaaat ctacgcaatt tctttatttc 4441 tgttttacgt gcaaataatt ttgatatggt aggttctaac ccttccataa ttcagaagta 4501 taatccaaac aatcaggatt atattgatga attgccatca tctgataatc aggaatatga 4561 tgataattcc gctccttctg gtggtttctt tgttccgcaa aatgataatg ttactcaaac 4621 ttttaaaatt aataacgttc gggcaaagga tttaatacga gttgtcgaat tgtttgtaaa 4681 gtctaatact tctaaatcct caaatgtatt atctattgac ggctctaatc tattagttgt 4741 tagtgctcct aaagatattt tagataacct tcctcaattc ctttcaactg ttgatttgcc 4801 aactgaccag atattgattg agggtttgat atttgaggtt cagcaaggtg atgctttaga 4861 tttttcattt gctgctggct ctcagcgtgg cactgttgca ggcggtgtta atactgaccg 4921 cctcacctct gttttatctt ctgctggtgg ttcgttcggt atttttaatg gcgatgtttt 4981 agggctatca gttcgcgcat taaagactaa tagccattca aaaatattgt ctgtgccacg 5041 tattcttacg ctttcaggtc agaagggttc tatctctgtt ggccagaatg tcccttttat 5101 tactggtcgt gtgactggtg aatctgccaa tgtaaataat ccatttcaga cgattgagcg 5161 tcaaaatgta ggtatttcca tgagcgtttt tcctgttgca atggctggcg gtaatattgt 5221 tctggatatt accagcaagg ccgatagttt gagttcttct actcaggcaa gtgatgttat 5281 tactaatcaa agaagtattg ctacaacggt taatttgcgt gatggacaga ctcttttact 5341 cggtggcctc actgattata aaaacacttc tcaggattct ggcgtaccgt tcctgtctaa 5401 aatcccttta atcggcctcc tgtttagctc ccgctctgat tctaacgagg aaagcacgtt 5461 atacgtgctc gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg 5521 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 5581 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 5641 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 5701 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 5761 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 5821 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggaa ccaccatcaa 5881 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg 5941 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct 6001 ggatccaagc ttgcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 6061 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 6121 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 6181 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 6241 gatgctgaag atcagttggg cgcactagtg ggttacatcg aactggatct caacagcggt 6301 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 6361 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 6421 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 6481 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 6541 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 6601 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 6661 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 6721 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 6781 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 6841 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 6901 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 6961 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 7021 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 7081 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactgt 7141 acgtaagacc cccaagcttg tcgactgaat ggcgaatggc gctttgcctg gtttccggca 7201 ccagaagcgg tgccggaaag ctggctggag tgcgatcttc ctgacgctcg agcgcaacgc 7261 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 7321 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 7381 tgattacgcc aagctttgga gccttttttt tggagatttt caacatgaaa tacctattgc 7441 ctacggcagc cgctggattg ttattactcg cGGCCcagcc GGCCatggcc gaagttcaat 7501 tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt tcttgcgctg 7561 cttccggatt cactttctct tcgtacgcta tgtcttgggt tcgccaagct cctggtaaag 7621 gtttggagtg ggtttctgct atctctggtt ctggtggcag tacttactat gctgactccg 7681 ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac ttgcagatga 7741 acagcttaag ggctgaggac actgcagtct actattgcgc taaagcctat cgtccttctt 7801 atcatgacat atggggtcaa ggtactatgg tcaccgtctc tagtgcctcc accaagggcc 7861 catcggtctt cccgctagca ccctcctcca agagcacctc tgggggcaca gcggccctgg 7921 gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc 7981 tgaccagcgg cgtccacacc ttcccggctg tcctacagtc ctcaggactc tactccctca 8041 gcagcgtagt gaccgtgccc tccagcagct tgggcaccca gacctacatc tgcaacgtga 8101 atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct tgtgcggccg 8161 cacatcatca tcaccatcac ggggccgcag aacaaaaact catctcagaa gaggatctga 8221 atggggccgc agaggctagc tctgctagtg gcgacttcga ctacgagaaa atggctaatg 8281 ccaacaaagg cgccatgact gagaacgctg acgagaatgc tttgcaaagc gatgccaagg 8341 gtaagttaga cagcgtcgcg accgactatg gcgccgccat cgacggcttt atcggcgatg 8401 tcagtggttt ggccaacggc aacggagcca ccggagactt cgcaggttcg aattctcaga 8461 tggcccaggt tggagatggg gacaacagtc cgcttatgaa caactttaga cagtaccttc 8521 cgtctcttcc gcagagtgtc gagtgccgtc cattcgtttt cggtgccggc aagccttacg 8581 agttcagcat cgactgcgat aagatcaatc ttttccgcgg cgttttcgct ttcttgctat 8641 acgtcgctac tttcatgtac gttttcagca ctttcgccaa tattttacgc aacaaagaaa 8701 gctagtgatc tcctaggaag cccgcctaat gagcgggctt tttttttctg gtatgcatcc 8761 tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat 8821 ctacaccaac gtgacctatc ccattacggt caatccgccg tttgttccca cggagaatcc 8881 gacgggttgt tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac 8941 gcgaattatt tttgatggcg ttcctattgg ttaaaaaatg agctgattta acaaaaattt 9001 aatgcgaatt ttaacaaaat attaacgttt acaatttaaa tatttgctta tacaatcttc 9061 ctgtttttgg ggcttttctg attatcaacc ggggtacata tgattgacat gctagtttta 9121 cgattaccgt tcatcgattc tcttgtttgc tccagactct caggcaatga cctgatagcc 9181 tttgtagatc tctcaaaaat agctaccctc tccggcatta atttatcagc tagaacggtt 9241 gaatatcata ttgatggtga tttgactgtc tccggccttt ctcacccttt tgaatcttta 9301 cctacacatt actcaggcat tgcatttaaa atatatgagg gttctaaaaa tttttatcct 9361 tgcgttgaaa taaaggcttc tcccgcaaaa gtattacagg gtcataatgt ttttggtaca 9421 accgatttag ctttatgctc tgaggcttta ttgcttaatt ttgctaattc tttgccttgc 9481 ctgtatgatt tattggatgt t

TABLE 35 DNA sequence of pMID21: 5957 bp (SEQ ID NO: 895)    1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt   61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt  121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat  181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt  241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg  301 ctgaagatca gttgggtgcc cgagtgggtt acatcgaact ggatctcaac agcggtaaga  361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc  421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac  481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg  541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca  601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg  661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg  721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg  781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag  841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg  901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct  961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1561 cgtgcataca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2221 accatgatta cgccaagctt tggagccttt tttttggaga ttttcaacgt gaaaaaatta 2281 ttattcgcaa ttcctttagt tgttcctttc tattctcaca gtgcacaggt ccaactgcag 2341 gagctcgaga tcaaacgtgg aactgtggct gcaccatctg tcttcatctt cccgccatct 2401 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 2461 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 2521 agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 2581 agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 2641 agttcaccgg tgacaaagag cttcaacagg ggagagtgtt aataaggcgc gcctaaccat 2701 ctatttcaag gaacagtctt aatgaaaaag cttttattca tgatcccgtt agttgtaccg 2761 ttcgtggccc agccggcctc tgctgaagtt caattgttag agtctggtgg cggtcttgtt 2821 cagcctggtg gttctttacg tctttcttgc gctgcttccg gagcttcaga tctgtttgcc 2881 tttttgtggg gtggtgcaga tcgcgttacg gagatcgacc gactgcttga gcaaaagcca 2941 cgcttaactg ctgatcaggc atgggatgtt attcgccaaa ccagtcgtca ggatcttaac 3001 ctgaggcttt ttttacctac tctgcaagca gcgacatctg gtttgacaca gagcgatccg 3061 cgtcgtcagt tggtagaaac attaacacgt tgggatggca tcaatttgct taatgatgat 3121 ggtaaaacct ggcagcagcc aggctctgcc atcctgaacg tttggctgac cagtatgttg 3181 aagcgtaccg tagtggctgc cgtacctatg ccatttgata agtggtacag cgccagtggc 3241 tacgaaacaa cccaggacgg cccaactggt tcgctgaata taagtgttgg agcaaaaatt 3301 ttgtatgagg cggtgcaggg agacaaatca ccaatcccac aggcggttga tctgtttgct 3361 gggaaaccac agcaggaggt tgtgttggct gcgctggaag atacctggga gactctttcc 3421 aaacgctatg gcaataatgt gagtaactgg aaaacaccgg caatggcctt aacgttccgg 3481 gcaaataatt tctttggtgt accgcaggcc gcagcggaag aaacgcgtca tcaggcggag 3541 tatcaaaacc gtggaacaga aaacgatatg attgttttct caccaacgac aagcgatcgt 3601 cctgtgcttg cctgggatgt ggtcgcaccc ggtcagagtg ggtttattgc tcccgatgga 3661 acagttgata agcactatga agatcagctg aaaatgtacg aaaattttgg ccgtaagtcg 3721 ctctggttaa cgaagcagga tgtggaggcg cataaggagt tctagagaca actctaagaa 3781 tactctctac ttgcagatga acagcttaag tctgagcatt cggtccgggc aacattctcc 3841 aaactgacca gacgacacaa acggcttacg ctaaatcccg cgcatgggat ggtaaagagg 3901 tggcgtcttt gctggcctgg actcatcaga tgaaggccaa aaattggcag gagtggacac 3961 agcaggcagc gaaacaagca ctgaccatca actggtacta tgctgatgta aacggcaata 4021 ttggttatgt tcatactggt gcttatccag atcgtcaatc aggccatgat ccgcgattac 4081 ccgttcctgg tacgggaaaa tgggactgga aagggctatt gccttttgaa atgaacccta 4141 aggtgtataa cccccagcag ctagccatat tctctcggtc accgtctcaa gcgcctccac 4201 caagggccca tcggtcttcc cgctagcacc ctcctccaag agcacctctg ggggcacagc 4261 ggccctgggc tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc 4321 aggcgccctg accagcggcg tccacacctt cccggctgtc ctacagtcta gcggactcta 4381 ctccctcagc agcgtagtga ccgtgccctc ttctagcttg ggcacccaga cctacatctg 4441 caacgtgaat cacaagccca gcaacaccaa ggtggacaag aaagttgagc ccaaatcttg 4501 tgcggccgca catcatcatc accatcacgg ggccgcagaa caaaaactca tctcagaaga 4561 ggatctgaat ggggccgcag aggctagttc tgctagtaac gcgtcttccg gtgattttga 4621 ttatgaaaag atggcaaacg ctaataaggg ggctatgacc gaaaatgccg atgaaaacgc 4681 gctacagtct gacgctaaag gcaaacttga ttctgtcgct actgattacg gtgctgctat 4741 cgatggtttc attggtgacg tttccggcct tgctaatggt aatggtgcta ctggtgattt 4801 tgctggctct aattcccaaa tggctcaagt cggtgacggt gataattcac ctttaatgaa 4861 taatttccgt caatatttac cttccctccc tcaatcggtt gaatgtcgcc cttttgtctt 4921 tggcgctggt aaaccatatg aattttctat tgattgtgac aaaataaact tattccgtgg 4981 tgtctttgcg tttcttttat atgttgccac ctttatgtat gtattttcta cgtttgctaa 5041 catactgcgt aataaggagt cttaatgaaa cgcgtgatga gaattcactg gccgtcgttt 5101 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 5161 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 5221 tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg 5281 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag 5341 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc 5401 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 5461 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 5521 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 5581 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 5641 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta 5701 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 5761 gtttacaatt ttatggtgca gtctcagtac aatctgctct gatgccgcat agttaagcca 5821 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 5881 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 5941 atcaccgaaa cgcgcga

TABLE 36 pM21J containing IIIss::A27::Ckappa Number of bases 5225 (SEQ ID NO: 921) GACGAAAGGG CCTCGTGATA CGCCTATTTT TATAGGTTAA TGTCATGATA ATAATGGTTT   60 CTTAGACGTC AGGTGGCACT TTTCGGGGAA ATGTGCGCGG AACCCCTATT TGTTTATTTT  120 TCTAAATACA TTCAAATATG TATCCGCTCA TGAGACAATA ACCCTGATAA ATGCTTCAAT  180 AATATTGAAA AAGGAAGAGT ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT  240 TTGCGGCATT TTGCCTTCCT GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG  300 CTGAAGATCA GTTGGGTGCC CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA  360 TCCTTGAGAG TTTTCGCCCC GAAGAACGTT TTCCAATGAT GAGCACTTTT AAAGTTCTGC  420 TATGTGGCGC GGTATTATCC CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC  480 ACTATTCTCA GAATGACTTG GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG  540 GCATGACAGT AAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA  600 ACTTACTTCT GACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG  660 GGGATCATGT AACTCGCCTT GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG  720 ACGAGCGTGA CACCACGATG CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG  780 GCGAACTACT TACTCTAGCT TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG  840 TTGCAGGACC ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG  900 GAGCCGGTGA GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT  960 CCCGTATCGT AGTTATCTAC ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC 1020 AGATCGCTGA GATAGGTGCC TCACTGATTA AGCATTGGTA ACTGTCAGAC CAAGTTTACT 1080 CATATATACT TTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA 1140 TCCTTTTTGA TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC CACTGAGCGT 1200 CAGACCCCGT AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG CGCGTAATCT 1260 GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG GATCAAGAGC 1320 TACCAACTCT TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA AATACTGTTC 1380 TTCTAGTGTA GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC 1440 TCGCTCTGCT AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG 1500 GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT 1560 CGTGCATACA GCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC CTACAGCGTG 1620 AGCTATGAGA AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT CCGGTAAGCG 1680 GCAGGGTCGG AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT 1740 ATAGTCCTGT CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG 1800 GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC CTGGCCTTTT 1860 GCTGGCCTTT TGCTCACATG TTCTTTCCTG CGTTATCCCC TGATTCTGTG GATAACCGTA 1920 TTACCGCCTT TGAGTGAGCT GATACCGCTC GCCGCAGCCG AACGACCGAG CGCAGCGAGT 1980 CAGTGAGCGA GGAAGCGGAA GAGCGCCCAA TACGCAAACC GCCTCTCCCC GCGCGTTGGC 2040 CGATTCATTA ATGCAGCTGG CACGACAGGT TTCCCGACTG GAAAGCGGGC AGTGAGCGCA 2100 ACGCAATTAA TGTGAGTTAG CTCACTCATT AGGCACCCCA GGCTTTACAC TTTATGCTTC 2160 CGGCTCGTAT GTTGTGTGGA ATTGTGAGCG GATAACAATT TCACACAGGA AACAGCTATG 2220 ACCATGATTA CGCCAAGCTT TGGAGCCTTT TTTTTGGAGA TTTTCAACAT GAAGAAACTG 2280 CTGTCTGCTA TCCCACTAGT TGTCCCTTTC TATTCTCATA GTGAAATCGT TCTGACCCAG 2340 TCCCCGGGGA CCCTGTCTCT GTCTCCGGGT GAACGTGCTA CGCTGAGCTG TCGTGCTTCT 2400 CAATCCGTTA GCTCCTCTTA TTTAGCTTGG TATCAGCAAA AGCCGGGTCA AGCTCCGCGG 2460 CTGTTGATCT ATGGTGCCTC TAGTCGTGCT ACTGGCATCC CTGATCGTTT CTCTGGCTCT 2520 GGCTCCGGAA CCGATTTCAC TCTGACCATT TCTCGTCTCG AGCCGGAAGA TTTCGCTGTC 2580 TACTATTGTC AACAGTATGG TTCTAGTCCG CTGACTTTCG GTGGCGGTAC CAAAGTCGAA 2640 ATCAAGCGTG GAACTGTGGC TGCACCATCT GTCTTCATCT TCCCGCCATC TGATGAGCAG 2700 TTGAAATCTG GAACTGCCTC TGTTGTGTGC CTGCTGAATA ACTTCTATCC CAGAGAGGCC 2760 AAAGTACAGT GGAAGGTGGA TAACGCCCTC CAATCGGGTA ACTCCCAGGA GAGTGTCACA 2820 GAGCAGGACA GCAAGGACAG CACCTACAGC CTCAGCAGCA CCCTGACTCT GTCCAAAGCA 2880 GACTACGAGA AACACAAAGT CTACGCCTGC GAAGTCACCC ATCAGGGCCT GAGTTCACCG 2940 GTGACAAAGA GCTTCAACAG GGGAGAGTGT TAATAAGGCG CGCCAATTTA ACCATCTATT 3000 TCAAGGAACA GTCTTAATGA AGAAGCTCCT CTTTGCTATC CCGCTCGTCG TTCCTTTTGT 3060 GGCCCAGCCG GCCATGGCCG AAGTTCAATT GTTAGAGTCT GGTGGCGGTC TTGTTCAGCC 3120 TGGTGGTTCT TTACGTCTTT CTTGCGCTGC TTCCGGATTC ACTTTCTCTC GTTACAAGAT 3180 GAAGTGGGTT CGCCAAGCTC CTGGTAAAGG TTTGGAGTGG GTTTCTGTTA TCTATCCTTC 3240 TGGTGGCGGT ACTGGTTATG CTGACTCCGT TAAAGGTCGC TTCACTATCT CTAGAGACAA 3300 CTCTAAGAAT ACTCTCTACT TGCAGATGAA CAGCTTAAGG GCTGAGGACA CTGCAGTCTA 3360 CTATTGTGCG AGAGTCAATT ACTATGATAG TAGTGGTTAC GGTCCTATAG CTCCTGGACT 3420 TGACTACTGG GGCCAGGGAA CCCTGGTCAC CGTCTCAAGC GCCTCCACCA AGGGTCCGTC 3480 GGTCTTCCCG CTAGCACCCT CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG 3540 CCTGGTCAAG GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC 3600 CAGCGGCGTC CACACCTTCC CGGCTGTCCT ACAGTCTAGC GGACTCTACT CCCTCAGCAG 3660 CGTAGTGACC GTGCCCTCTT CTAGCTTGGG CACCCAGACC TACATCTGCA ACGTGAATCA 3720 CAAGCCCAGC AACACCAAGG TGGACAAGAA AGTTGAGCCC AAATCTTGTG CGGCCGCACA 3780 TCATCATCAC CATCACGGGG CCGCAGAACA AAAACTCATC TCAGAAGAGG ATCTGAATGG 3840 GGCCGCAGAG GCTAGTTCTG CTAGTAACGC GTCTTCCGGT GATTTTGATT ATGAAAAGAT 3900 GGCAAACGCT AATAAGGGGG CTATGACCGA AAATGCCGAT GAAAACGCGC TACAGTCTGA 3960 CGCTAAAGGC AAACTTGATT CTGTCGCTAC TGATTACGGT GCTGCTATCG ATGGTTTCAT 4020 TGGTGACGTT TCCGGCCTTG CTAATGGTAA TGGTGCTACT GGTGATTTTG CTGGCTCTAA 4080 TTCCCAAATG GCTCAAGTCG GTGACGGTGA TAATTCACCT TTAATGAATA ATTTCCGTCA 4140 ATATTTACCT TCCCTCCCTC AATCGGTTGA ATGTCGCCCT TTTGTCTTTG GCGCTGGTAA 4200 ACCATATGAA TTTTCTATTG ATTGTGACAA AATAAACTTA TTCCGTGGTG TCTTTGCGTT 4260 TCTTTTATAT GTTGCCACCT TTATGTATGT ATTTTCTACG TTTGCTAACA TACTGCGTAA 4320 TAAGGAGTCT TAATGAAACG CGTGATGAGA ATTCACTGGC CGTCGTTTTA CAACGTCGTG 4380 ACTGGGAAAA CCCTGGCGTT ACCCAACTTA ATCGCCTTGC AGCACATCCC CCTTTCGCCA 4440 GCTGGCGTAA TAGCGAAGAG GCCCGCACCG ATCGCCCTTC CCAACAGTTG CGCAGCCTGA 4500 ATGGCGAATG GCGCCTGATG CGGTATTTTC TCCTTACGCA TCTGTGCGGT ATTTCACACC 4560 GCATACGTCA AAGCAACCAT AGTACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT 4620 GGTGGTTACG CGCAGCGTGA CCGCTACACT TGCCAGCGCC TTAGCGCCCG CTCCTTTCGC 4680 TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCTTTCCC CGTCAAGCTC TAAATCGGGG 4740 GCTCCCTTTA GGGTTCCGAT TTAGTGCTTT ACGGCACCTC GACCCCAAAA AACTTGATTT 4800 GGGTGATGGT TCACGTAGTG GGCCATCGCC CTGATAGACG GTTTTTCGCC CTTTGACGTT 4860 GGAGTCCACG TTCTTTAATA GTGGACTCTT GTTCCAAACT GGAACAACAC TCAACTCTAT 4920 CTCGGGCTAT TCTTTTGATT TATAAGGGAT TTTGCCGATT TCGGTCTATT GGTTAAAAAA 4980 TGAGCTGATT TAACAAAAAT TTAACGCGAA TTTTAACAAA ATATTAACGT TTACAATTTT 5040 ATGGTGCAGT CTCAGTACAA TCTGCTCTGA TGCCGCATAG TTAAGCCAGC CCCGACACCC 5100 GCCAACACCC GCTGACGCGC CCTGACGGGC TTGTCTGCTC CCGGCATCCG CTTACAGACA 5160 AGCTGTGACC GTCTCCGGGA GCTGCATGTG TCAGAGGTTT TCACCGTCAT CACCGAAACG 5220 CGCGA 5225

TABLE 40 pLCSK23 (SEQ ID NO: 896)    1 GACGAAAGGG CCTGCTCTGC CAGTGTTACA ACCAATTAAC CAATTCTGAT TAGAAAAACT   61 CATCGAGCAT CAAATGAAAC TGCAATTTAT TCATATCAGG ATTATCAATA CCATATTTTT  121 GAAAAAGCCG TTTCTGTAAT GAAGGAGAAA ACTCACCGAG GCAGTTCCAT AGGATGGCAA  181 GATCCTGGTA TCGGTCTGCG ATTCCGACTC GTCCAACATC AATACAACCT ATTAATTTCC  241 CCTCGTCAAA AATAAGGTTA TCAAGTGAGA AATCACCATG AGTGACGACT GAATCCGGTG  301 AGAATGGCAA AAGCTTATGC ATTTCTTTCC AGACTTGTTC AACAGGCCAG CCATTACGCT  361 CGTCATCAAA ATCACTCGCA TCAACCAAAC CGTTATTCAT TCGTGATTGC GCCTGAGCGA  421 GACGAAATAC GCGATCGCTG TTAAAAGGAC AATTACAAAC AGGAATTGAA TGCAACCGGC  481 GCAGGAACAC TGCCAGCGCA TCAACAATAT TTTCACCTGA ATCAGGATAT TCTTCTAATA  541 CCTGGAATGC TGTTTTCCCG GGGATCGCAG TGGTGAGTAA CCATGCATCA TCAGGAGTAC  601 GGATAAAATG CTTGATGGTC GGAAGAGGCA TAAATTCCGT CAGCCAGTTT AGTCTGACCA  661 TCTCATCTGT AACATCATTG GCAACGCTAC CTTTGCCATG TTTCAGAAAC AACTCTGGCG  721 CATCGGGCTT CCCATACAAT CGATAGATTG TCGCACCTGA TTGCCCGACA TTATCGCGAG  781 CCCATTTATA CCCATATAAA TCAGCATCCA TGTTGGAATT TAATCGCGGC CTCGAGCAAG  841 ACGTTTCCCG TTGAATATGG CTCATAACAC CCCTTGTATT ACTGTTTATG TAAGCAGACA  901 GTTTTATTGT TCATGATGAT ATATTTTTAT CTTGTGCAAT GTAACATCAG AGATTTTGAG  961 ACACAACGTG GCTTTCCCCC CCCCCCCCTG CAGGTCTCGG GCTATTCCTG TCAGACCAAG 1021 TTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT TTAATTTAAA AGGATCTAGG 1081 TGAAGATCCT TTTTGATAAT CTCATGACCA AAATCCCTTA ACGTGAGTTT TCGTTCCACT 1141 GAGCGTCAGA CCCCGTAGAA AAGATCAAAG GATCTTCTTG AGATCCTTTT TTTCTGCGCG 1201 TAATCTGCTG CTTGCAAACA AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC 1261 AAGAGCTACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG ATACCAAATA 1321 CTGTTCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA GAACTCTGTA GCACCGCCTA 1381 CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC 1441 TTACCGGGTT GGACTCAAGA CGATAGTTAC CGGATAAGGC GCAGCGGTCG GGCTGAACGG 1501 GGGGTTCGTG CATACAGCCC AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC 1561 AGCGTGAGCT ATGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC AGGTATCCGG 1621 TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA AACGCCTGGT 1681 ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA GCGTCGATTT TTGTGATGCT 1741 CGTCAGGGGG GCGGAGCCTA TGGAAAAACG CCAGCAACGC GGCCTTTTTA CGGTTCCTGG 1801 CCTTTTGCTG GCCTTTTGCT CACATGTTCT TTCCTGCGTT ATCCCCTGAT TCTGTGGATA 1861 ACCGTATTAC CGCCTTTGAG TGAGCTGATA CCGCTCGCCG CAGCCGAACG ACCGAGCGCA 1921 GCGAGTCAGT GAGCGAGGAA GCGGAAGAGC GCCCAATACG CAAACCGCCT CTCCCCGCGC 1981 GTTGGCCGAT TCATTAATGC AGCTGGCACG ACAGGTTTCC CGACTGGAAA GCGGGCAGTG 2041 AGCGCAACGC AATTAATGTG AGTTAGCTCA CTCATTAGGC ACCCCAGGCT TTACACTTTA 2101 TGCTTCCGGC TCGTATGTTG TGTGGAATTG TGAGCGGATA ACAATTTCAC ACAGGAAACA 2161 GCTATGACCA TGATTACGCC AAGCTTTGGA GCCTTTTTTT TGGAGATTTT CAACATGAAG 2221 AAGCTCCTCT TTGCTATCCC GCTCGTCGTT CCTTTTGTGG CCCAGCCGGC CATGGCCGAC 2281 ATCCAGATGA CCCAGTCTCC ATCCTCCCTG TCTGCATCTG TAGGAGACAG AGTCACCATC 2341 ACTTGCCGGG CAAGTCAGAG CATTAGCAGC TATTTAAATT GGTATCAGCA GAAACCAGGG 2401 AAAGCCCCTA AGCTCCTGAT CTATGCTGCA TCCAGTTTGC AAAGTGGGGT CCCATCAAGG 2461 TTCAGTGGCA GTGGATCTGG GACAGATTTC ACTCTCACCA TCAGCAGTCT GCAACCTGAA 2521 GATTTTGCAA CTTACTACTG TCAACAGAGT TACAGTACCC CTTTCACTTT CGGCCCTGGG 2581 ACCAAAGTGG ATATCAAACG TGGtACcGTG GCTGCACCAT CTGTCTTCAT CTTCCCGCCA 2641 TCTGATGAGC AGTTGAAATC TGGAACTGCC TCTGTTGTGT GCCTGCTGAA TAACTTCTAT 2701 CCCAGAGAGG CCAAAGTACA GTGGAAGGTG GATAACGCCC TCCAATCGGG TAACTCCCAG 2761 GAGAGTGTCA CAGAGCAGGA CAGCAAGGAC AGCACCTACA GCCTCAGCAG CACCCTGACG 2821 CTGAGCAAAG CAGACTACGA GAAACACAAA GTCTACGCCT GCGAAGTCAC CCATCAGGGC 2881 CTGAGTTCAC CGGTGACAAA GAGCTTCAAC AGGGGAGAGT GTGCGGCCGC TGGTAAGCCT 2941 ATCCCTAACC CTCTCCTCGG TCTCGATTCT ACGTGATAAC TTCACCGGTC AACGCGTGAT 3001 GAGAATTCAC TGGCCGTCGT TTTACAACGT CGTGACTGGG AAAACCCTGG CGTTACCCAA 3061 CTTAATCGCC TTGCAGCACA TCCCCCTTTC GCCAGCTGGC GTAATAGCGA AGAGGCCCGC 3121 ACCGATCGCC CTTCCCAACA GTTGCGCAGC CTGAATGGCG AATGGCGCCT GATGCGGTAT 3181 TTTCTCCTTA CGCATCTGTG CGGTATTTCA CACCGCATAC GTCAAAGCAA CCATAGTCTC 3241 AGTACAATCT GCTCTGATGC CGCATAGTTA AGCCAGCCCC GACACCCGCC AACACCCGCT 3301 GACGCGCCCT GACAGGCTTG TCTGCTCCCG GCATCCGCTT ACAGACAAGC TGTGACCGTC 3361 TCCGGGAGCT GCATGTGTCA GAGGTTTTCA CCGTCATCAC CGAAACGCGC GA

Example 4: Dobbling of CDRs

The following examples exemplify the use of dobbling in constructing synthetic libraries. The parental 3-23 heavy chain (HC) is diversified in CDR1, 2, and 3. This diversity is combined with a synthetically diversified A27 light chain (LC). The diversity will be as follows:

Example 4.1 HC CDR1

The following dobbling diversity allows 5,832 variants. See Table 50. At position 31, Ser is the germline (GL) amino-acid type. Hence we make Ser three times more likely then the other types. Since 18 types are allowed, Ser will be allowed 15% of the time and all the others are allowed at 5%. Thus, if there is no selection for the AA type at 31, we are more likely to isolate an Ab with Ser. Similarly, at 33 the GL AA type is Ala and we make Ala 3 times as likely (15%) as all the others (5%). At 35 Ser is the GL AA type and we make it three times as likely as the others. At all three positions, we have excluded Cys and Met. We exclude Cys because we do not want gratuitus disulfides or exposed unpaired cysteines that could adversely affect the solubility and reactivity of the Ab. We exclude Met because exposed methionines side groups are subject to oxidation which can alter binding properties and shelf life. We could make the germline amino-acid type 2, 3, 4, 5, 6, 8, or 10 times more likely than the other AA types.

TABLE 50 Diversity for CDR1 in 3-23 Position Parental AA Allowed 31 S (three-times more ADEFGHKLNPQRSTVWY likely as the others) 33 A (3-X more likely) ADEFGHKLNPQRSTVWY 35 S (3-X more likely) ADEFGHKLNPQRSTVWY

Throughout this disclosure, the shown “Allowed” amino acids are the amino acids that can be used at a given position. For example, in Table 50, at position 31, allowed amino acids “ADEFGHKLNPQRSTVWY” are shown. This indicates that amino acids A, D, E, F, G, H, K, L, N, P, Q, R, S, T, V, W, and Y are all allowed at position 31.

Example 4.2: HC CDR2

In CDR2, we allow diversity at positions 50, 52, 52a, 56, and 58. At 50, 52, 56, and 58 we allow all amino-acid types except Cys and Met and we make the GL AA types more likely by three fold. We could make the GL AA type 2, 3, 4, 5, 6, 8, or 10 times more likely than the other AA types.

TABLE 51 HC CDR2: Diversity = 419,904 Position Parental AA Allowed 50  A (3-X more likely) ADEFGHKLNPQRSTVWY 52  S (3-X more likely) ADEFGHKLNPQRSTVWY 52a G (3-X more likely) GPSY 56  S (3-X more likely) ADEFGHKLNPQRSTVWY 58  Y (3-X more likely) ADEFGHKLNPQRSTVWY

Combined CDR1 and CDR2 diversity=2.45 E 9

Example 4.3 HC CDR3, Lengths 3, 4, 5

Very short CDR3 can be made by dobbling. Table 7 shows several parental sequences for CDR3 length 3. At 94 many VH3s have Arg and we have allowed this change, but Lys is made 3-X as likely. At 95, F is found at this position in JH1. We also allow Ser, Tyr, Asp, and Arg to allow small, large, plus charge, and minus charge. At 96, JH1 has Q. Since Q is very similar to Glu, we allow Glu as an acidic alternative plus Arg, Ser, Tyr, and Leu. At 97, His is the germline AA from JH1. We allow minus charge (D), plus charge (R), small polar (S), large hydrophobic (Y), and aliphatic (L). The parental sequence makes up 4.5% of the library, but this is combined with a large diversity in CDR1 and CDR2. The dobbling allows 360 sequences in all. The least likely sequences occur at 1 in 1792. The most likely (parental) sequence occurs about 1 in 22.

TABLE 60 A dobbled HC CDR3 of length 3 (V-3JH1 of Table 7) (‘KFQH’ disclosed as SEQ ID NO: 951) Parental amino Position acid (source) Allowed  94 K (VH 3-23) KR (3:1)  95 F (JH1) FSYDR (3:1:1:1:1)  96 Q (JH1) QERSYL (3:1:1:1:1:1)  97 H (JH1) HDRSYL (3:1:1:1:1:1) 103 W (JH1) W

Table 61 shows a dobbled HC CDR3 of length 3. Here K94 is fixed as is W103. We have made the “parental” D segment amino acid five times as likely as the other allowed AA types.

TABLE 61 A dobbled HC CDR3 of length 3 from a D  fragment (V-3D1-1.1.2-JH1 of Table 7). (‘KTTG’ disclosed as SEQ ID NO: 952) Position Parental Allowed 94 K (V 3-23) K 95 T (D1-1.1.2) TYRDL (5:1:1:1:1) 96 T (D1-1.1.2) TYRDL (5:1:1:1:1) 97 G (D1-1.1.2) GSYRDL (5:1:1:1:1:1) 103 W (JH1) W

In this example (Table 62, using V-4JH2 from Table 8), 94 is fixed as Lys. At 95, JH2 has Tyr and we have allowed Ser, Asp, Arg, and Leu so that size, charge, and hydrophobicity can alter to suit the antigen. JH2 has Phe at 96 and we have allowed Ser, Tyr, Asp, Arg, and Leu. At 97, JH2 has Asp and we have allowed Arg, Ser, Tyr, and Leu. At 98, JH2 has Leu and we have allowed Ser, Tyr, Asp, and Arg. This pattern allows 750 distinct sequences, of which the parental is the most likely (1 in 18). The least likely sequences occur at 1 in 4608 or 256 times less likely than the most likely.

TABLE 62 HC CDR3 length 4 from JH2 (V-4JH2 in Table 7) (‘KYFDL’ disclosed as SEQ ID NO: 953) Position Parental AA (source) Allowed 94 K (VH 3-23) K 95 Y (JH2) YSDRL (4:1:1:1:1) 96 F (JH2) FSYDRL (4:1:1:1:1:1) 97 D (JH2) DRSYL (4:1:1:1:1) 98 L (JH2) LSYDR (4:1:1:1:1) 103 W (JH2) W

In Table 63, there is a dobbling of V-4D3-10.1a-JH2 from Table 8. At 94, we allow Lys and Arg with Lys (the parental) four times as likely as Arg. At 95, D3-10.1a (i.e., D3-10 in the first reading frame and starting a AA 1) has Leu; we allow SYDR as well with Leu 4-X as likely as each of the other AA types. At 96, D3-10.1a has Leu again and we allow the same menu. At 97, D3-10.1a has Trp and we allow Ser, Tyr, Asp, and Arg with Trp 4-X as likely. At 98, D3-10.1a has Phe and we allow Ser, Tyr, Asp, and Arg as well.

TABLE 63 HC CDR3 of length four from V-4D3-10.1a in Table 8 (‘KLLWF’ disclosed as SEQ ID NO: 954) Position Parental AA (source) Allowed 94 K (VH 3-23) KR (4:1) 95 L (D3-10.1a) LSYDR (4:1:1:1:1) 96 L (D3-10.1a) LSYDR (4:1:1:1:1) 97 W (D3-10.1a) WSYDR (4:1:1:1:1) 98 F (D3-10.1a) FSYDR (4:1:1:1:1) 103 W W

Example 4.4: HC CDR3 Length 10 to 20

HC CDR3

Two sublibraries, both with CDR3 of length 16:

TABLE 52 Library 1: Diversity = 5 E 11, the “parental” sequence occurs at 1 in 1.5 E6. (‘KYYYDSSGYYYAEYFQHW’ disclosed as SEQ ID NO: 955) Position “Parental” AA (source) Allowed  94 K (3-X more likely) (3-23) KR (3:1)  95 Y (3-X more likely) (D2-21(2)) YSRDL (3:1:1:1:1)  96 Y (3-X more likely) (D2-21(2)) YSRDL (3:1:1:1:1)  97 Y (3-X more likely) (D2-21(2)) YSRDL (3:1:1:1:1)  98 D (3-X more likely) (D2-21(2)) DYSRL (3:1:1:1:1)  99 S (3-X more likely) (D2-21(2)) SYRDL (3:1:1:1:1) 100 S (3-X more likely) (D2-21(2)) SYRDL (3:1:1:1:1) 101 G (3-X more likely) (D2-21(2)) GASYRDL (3:1:1:1:1:1:1) 102 Y (3-X more likely) (D2-21(2)) YSRDL (3:1:1:1:1) 102a Y (3-X more likely) (D2-21(2)) YSRDL (3:1:1:1:1) 102b Y (3-X more likely) (D2-21(2)) YSRDL (3:1:1:1:1) 102c A (3-X more likely) (JH1) ASYRD (3:1:1:1:1) 102d E (3-X more likely) (JH1) ERSYL (3:1:1:1:1) 102e Y (3-X more likely) (JH1) YSRDL (3:1:1:1:1) 102f F (3-X more likely) (JH1) FYSRD (3:1:1:1:1) 102g Q (3-X more likely) (JH1) QERSY (3:1:1:1:1) 102h H (3-X more likely) (JH1) HERSYL (3:1:1:1:1:1) 103 W (JH1, fixed) W

TABLE 53 Library 2: CDR3 length 16; Diversity is 3.0 E 10 and the parental sequence occurs once in 3.7 E 5. (‘KGYCSSTSCYTAEYFQHW’ disclosed as SEQ ID NO: 956) Position “Parental” AA (source) Allowed  94 K (3-X more likely) (3-23) KR (3:1)  95 G (3-X more likely) (D2-2(2)) GSYDRL (3:1:1:1:1:1)  96 Y (3-X more likely) (D2-2(2)) YSDRL (3:1:1:1:1)  97 C (fixed) (D2-2(2)) C  98 S (3-X more likely) (D2-2(2)) SYRDL (3:1:1:1:1)  99 S (3-X more likely) (D2-2(2)) SYRDL (3:1:1:1:1) 100 T (3-X more likely) (D2-2(2)) TYRDL (3:1:1:1:1) 101 S (3-X more likely) (D2-2(2)) SYRDL (3:1:1:1:1) 102 C (fixed) (D2-2(2)) C 102a Y (3-X more likely) (D2-2(2)) YSDRL (3:1:1:1:1) 102b T (3-X more likely) (D2-2(2)) TYRDL (3:1:1:1:1) 102c A (3-X more likely) (JH1) ASYDRL (3:1:1:1:1:1) 102d E (3-X more likely) (JH1) ERSYL (3:1:1:1:1) 102e Y (3-X more likely) (JH1) YSDRL (3:1:1:1:1) 102f F (3-X more likely) (JH1) FYSRDL (3:1:1:1:1:1) 102g Q (3-X more likely) (JH1) QERSYL (3:1:1:1:1:1) 102h H (3-X more likely) (JH1) HDRSYL (3:1:1:1:1:1) 103 W ((JH1)) W

Table 65 shows a dobbling variegation of SEQ ID NO:898. The total diversity allowed is 2.1 E 13. A synthesis that produces 1. E 8, 3. E 8, 5. E 8, 1. E 9, or 5. E 9 will sample the diversity adequately. The design of SEQ ID NO:898 was discussed above. In dobbling SEQ ID NO:898, is to allow the parental AA type at three-fold above other AA types at most positions. At positions where the parental is Tyr, then we use Tyr and Ser at equal amounts with Leu at one half that frequency. The Cys residues are fixed. Each parental AA type is allowed to go to one of Arg, Asp, Ser, Tyr, or Leu (Leu might be omitted if the parental is hydrophobic, such as Phe). The parental sequence will occur once in 1. E 8 members. The least likely sequences will occur once in 9.5 E 16. It is not important that the library actually contain the parental sequence, only that it contains many sequences that resemble the parent. Thus, a library that contains 1. E 7, 5. E7, 1.E8, 3. E8, 1. E 9, or 5. E 9, when combined with diversity in HC CDR1, HC CDR2, LC CDR1, LC CDR2, and LC CDR3 will provide a library that will contain many valuable Abs.

TABLE 65 Dobbling of Design 1 with SEQ ID NO:898 as parent (‘KDYGYCSSTSCYTYGYSYAEYFQHW’  disclosed as SEQ ID NO: 957) Position Parental (source) Allowed  94 K (VH 3-23) K  95 D (No source) DSYL (3:1:1:1)  96 Y (No source) YSL (2:2:1)  97 G (D2-2.2) GSYDRL (3:1:1:1:1:1)  98 Y (D2-2.2) YSL (2:2:1)  99 C (D2-2.2) C 100 S (D2-2.2) SYDRL (3:1:1:1:1) 101 S (D2-2.2) SYDRL (3:1:1:1:1) 102 T (D2-2.2) TYDRL (3:1:1:1:1) 102a S (D2-2.2) SYDRL (3:1:1:1:1) 102b C (D2-2.2) C 102c Y (D2-2.2) YSL (2:2:1) 102d T (D2-2.2) TYDRL (3:1:1:1:1) 102e Y (No source) YDSL (3:1:1:1) 102f G (No source) GSYRD (3:1:1:1:1) 102g Y (No source) YSL (2:2:1) 102h S (No source) SYDRL (3:1:1:1) 102i Y (No source) YSL (2:2:1) 102j A (JH1) ASYDR (3:1:1:1:1) 102k E (JH1) ERSYL (3:1:1:1:1) 1021 Y (JH1) YSL (2:2:1) 102m F (JH1) FSYDR (3:1:1:1:1) 102n Q (JH1) QYSDRL (3:1:1:1:1:1) 102p H (JH1) HSYDRL (3:1:1:1:1:1) 103 W (JH1, FR4) W

Example 4.5 Dobbling of yycakGSGYCSGGSCYSFDYwgqgtivtvss (SEQ ID NO:931)

Table 80 shows the dobbling of SEQ ID NO:931, an example of an HC CDR3 of length 15. Position 94 is part of FR3 and is held constant. Positions 95 and 96 have “parental” amino-acid types picked from the highly used set of (YGDRS) and are G95 and S96. The next ten positions are taken from D2-15.2 (a moderately highly used D segment containing a disulfide-closed loop). The final three positions are from the JH4 positions 100, 101, and 102 as shown in Table 3. At each position, we make the parental amino-acid type three times more likely than the other allowed types. The Cys residues are fixed. At 102e, Phe is three times more likely as are YGSRD (i.e., Phe is three times more likely as are any of amino acids Y, G, S, R, or D). The diversity allowed is 1.46 E 9. The parental sequence is expected at 1 in 6.9 E 4. Each of the singly substituted sequences is about ⅓ as likely; the doubly substituted ones are 1/9 as likely and so on. The sequences that are composed entirely of other AA types occur at only 1 in 1.1 E 11.

Each of the other sequences in Table 21 can be dobbled in the same way.

TABLE 80 Dobbling of yycakGSGYCSGGSCYSFDYwgqgtivtvss (SEQ ID NO: 931) (‘KGSGYCSGGSCYSFDYW’ disclosed as SEQ ID NO: 958) Position Parental (source) Allowed  94 K (VH 3-23) K  95 G (No source) GYSRD (3:1:1:1:1)  96 S (No source) SGYRD (3:1:1:1:1)  97 G (D2-15.2) GYSRD (3:1:1:1:1)  98 Y (D2-15.2) YGSRD (3:1:1:1:1)  99 C (D2-15.2) C 100 S (D2-15.2) SGYRD (3:1:1:1:1) 101 G (D2-15.2) GYSRD (3:1:1:1:1) 102 G (D2-15.2) GYSRD (3:1:1:1:1) 102a S (D2-15.2) SGYRD (3:1:1:1:1) 102b C (D2-15.2) C 102c Y (D2-15.2) YGSRD (3:1:1:1:1) 102d S (D2-15.2) SGYRD (3:1:1:1:1) 102e F (JH4) FYGSRD (3:1:1:1:1:1) 102f D (JH4) DGSRY (3:1:1:1:1) 102g Y (JH4) YGSRD (3:1:1:1:1) 103 W (JH4, FR4) W

Example 5: Synthetic Light Chain Diversity

To make whole antibodies, we need to combine a library of heavy chains with a library of light chains (LC). In natural Abs, it is often observed that HC does most of the binding and many libraries have given little attention to the LC or have obtained LC diversity from human donors. To have enough diversity to give good binders to almost any target, we have designed a diversification program that exceeds what the human immune system usually provides. Nevertheless, the program is designed to yield fully functional LC that have the same kind of changes as seen in natural Abs, only a few more. Vkappa III A27 was picked as the LC.

From a library that comprises donated kappa and lambda LCs, a collection of 1266 Abs were typed. Among VKIIIs, A27 is most often seen (Table 66) and pairs well with HC 3-23.

The CDRs of A27 contain 12, 7, and 9 amino acids. Putting diversity at all of these positions might not work well: a) there might be many unstable or non-functional members, and b) diversity at some positions might not help improve binding. We have reduced the number of variable positions from 28 to 16.

We have studied the 3D structure of 1QLR which has an A27 LC. The 1GLR structure is publicly available in the RCDB Protein Data Base. From this, the residues marked in Table 68 look useful to vary. The T56 is about 10 Å from a His in HC CDR3. Variation at 56 may be useful. G24 is only about 7 Å from an atom in HC CDR3. Germline is R24; thus, variation at 24 may be useful.

Table 69 shows a display cassette that we designed for use in pMID21. Thus, the restriction enzymes picked do not have other sites in pMID21. SpeI is in the iii signal sequence and allows the entire LC to be inserted or removed. XmaI, PpuMI, EcoO109I, and BlpI precede CDR1. SacII is in FR2, separating CDR1 from CDR2. Alternatively, an AvrII site could be inserted at the same position. BspEI and XhoI sites are in FR3 and a KpnI site is in FR4.

We gathered 155 A27 sequences and analyzed what happens in the CDRs. Table 70 shows the analysis. In Table 70, we show what is found in the Abs from our library and what we would put at each position.

TABLE 68 where to vary A27 22    3    3 5    5  89    9 45    0a   4 0    5  90    5 1QLR GASQSVS_NYLA DASSRAT QQYGSSPLT A27 RASQSVSSSYLA GASSRAT QQYGSSPLT  ** **** * * * * ****** GASQSVS is (SEQ ID NO: 922) NYLA (SEQ ID NO: 959) DASSRAT is (SEQ ID NO: 923) QQYGSSPLY is (SEQ ID NO: 924) QQYGSSPLT (SEQ ID NO: 966) RASQSVSSSYLA is (SEQ ID NO: 925) GASSRAT is (SEQ ID NO: 926)

Table 68 shows where the CDRs of A27 would be variegated.

TABLE 67 Compare VKIII AA seqs VK3 is (SEQ ID NO: 927) A27 is (SEQ ID NO: 960) A11 is (SEQ ID NO: 961) L2 is (SEQ ID NO: 962) L16 is (SEQ ID NO: 962) L6 is (SEQ ID NO: 963) L20 is (SEQ ID NO: 964) L25 is (SEQ ID NO: 965)  FR1....................CDR1........FR2............CDR2...FR3          1         2         3          4         5         6 1234567890123456789012345678901a23456789012345678901234567890 VK3 DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPA A27 E-------G-------------------------------------------------I-D A11 E----------------------G------------------L-------D-------I-D L2 E--M--------V------------------#N--------------------T----I-- L16 E--M--------V------------------#N--------------------T----I-- L6 E------------------------------#------------------D--N----I-- L20 E--------------------------G---#------------------D--N----I-- L25 E--M------------------------------S------------------T----I-- .............................CDR3.....FR4.........                                        1          7         8         9         0 1234567890123456789012345678901234567890123456789 VK3 RFSGSGSGTDFTLTISSLEPEDFAVYYCQQHYTTPPTFGQGTKVEIKRT A27 ----------------R-------------YGSS- A11 ----------------R-------------YGSS- L2 ---------E--------QS----------YNNW- L16 ---------E--------QS----------YNNW- L6 ------------------------------RSNW- L20 -------P----------------------RSNWH L25 ------------------Q-----------D-NL- VK3 differs from A27 by E1D, G9A, I58V, D60A, R77S. A27 and L6 differ by G9A(FR1), A31a(in CDR1), G50D(CDR2),  S53N(CDR2), G92S(CDR3), S93N(CDR3), S94W(CDR3) VK3 from U.S. Pat. No. 7, 264, 963.

CDR1

R24, A25, and S26 are too far from the combining site to help and were held constant. The side group of V29 is buried; this position was held constant as Val. At the other positions, we allowed Y or S and a charge flip-flop (RE or RD, depending on where the sample had more of E or D at the position in question) plus other types that were frequently seen. We used an Excel spread sheet to determine that this pattern of variegation would give the parental sequence at 0.8% if the “other” AAs were substituted at 5%, at 0.1% if the “other” AAs were substituted at 6.5%, and at 0.02% if “other” was at 9%. In the sample of 155, 17 have one AA deleted (including 1QLR); thus, we will arrange to have S30a deleted in ˜8% of the members.

CDR2

From inspection of 1QLR, we see that CDR2 is somewhat remote from the combining site. There have even been suggestions that we keep the residues in this CDR constant. Studying the 3D structure suggests that variegation at G50, S53, and T56 could be useful. S53 is the most variable in the sample of 155, but this does not prove that these changes are useful. In 1QLR, G50 has been mutated to R50. The side group of T56 is pointed toward HC CDR3 and is about 11 Å from an atom in HC CDR3.

CDR3

Q89 and Q90 are buried and nature does not vary them often; these residues are not varied. Y91 is packed against HC CDR3 and changes here would alter the combining site and do occur. At G92, φ=−80 and ψ=−15 so putting in a non-Gly is feasible; nature does it in 47/155 cases. S93 is very often varied or deleted. S94 is highly exposed and is highly varied. P95 is exposed and varied. L96 packs against HC CDR3: changes here will affect the binding site and do occur in nature. T97 is buried and has been held constant/the amino acid is not varied.

The parental sequence appears at 0.000246 or 1 in 4.06 E3. The allowed diversity is about 2.1 E 12. With two 8% deletions, 84.6% of the members will be full length, 7.4% will have short CDR1 and full-length CDR3, 7.4% will have full-length CDR1 and short CDR3, and 0.6% will have both deletions.

Other germlines were not in the sample.

TABLE 66 Distribution of VLs in 1266 selected LCs. Kappas Lambdas O12 VKI 313 1a VL1 9 O18 VKI 1 1e VL1 7 A20 VKI 26 1c VL1 55 A30 VKI 26 1g VL1 46 L14 VKI 2 1b VL1 1 118 L1 VKI 5 2c VL2 18 L15 VKI 1 2e VL2 23 L5 VKI 83 2a2 VL2 79 L8 VKI 10 2d VL2 1 121 L12 VKI 77 544 3r VL3 56 O11 VKII 4 3j VL3 4 A17 VKII 17 31 VL3 31 A19 VKII 31 52 3h VL3 22 113 A27 VKIII 155 4a VL4 1 1 L2 VKIII 31 5c VL5 1 1 L6 VKIII 88 6a VL6 8 8 L25 VKIII 16 290 10a VL10 6 6 B3 VKIV 12 12 Number of lambdas 368 Number of kappas 898 Total Abs in sample 1266

TABLE 69 A Display gene for A27 in pM21J. IIIsignal::A27::Ckappa The amino-acid sequence of Table 69 is (SEQ ID NO: 928). The DNA sequence of Table 69 is (SEQ ID NO: 929).  signal sequence--------------------------------------------    1   2   3   4   5   6   7   8   9  10  11  12  13  14  15    M   K   K   L   L   S   A   I   P   L   V   V   P   F   Y  1  |atg|aaG|aaA|ctg|ctg|tct|gct|atc|ccA|CTA|GTt|gtc|cct|ttc|tat|                                     SpeI.... Signal------- FR1-------------------------------------------   16  17  18  19  20  21  22  23  24  25  26  27  28  29  30    S   H   S   E1  I   V3  L   T5  Q   S7  P   G9  T   L   S12  46  |tct|cat|agt|gaa|atc|gtt|ctg|acc|cag|tcC|CCG|GGG|aCC|Ctg|tct|                                         XmaI....                                               PpuMI....                                               EcoO109I.(1/2)   FR1--------------------------------------- CDR1-----------   31  32  33  34  35  36  37  38  39  40  41  42  43  44  45   L13  S   P   G   E   R   A   T   L   S  C23 R24  A   S   Q  91  |ctg|tct|ccg|ggt|gaa|cgt|gct|acG|CTg|AGC|tgt|cgt|gct|tct|caa|                                 BlpI.....   CDR1--------------------------  FR2------------------------   46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   S28  V   S   S  S30a Y   L  A34  W   Y   Q   Q   K   P   G 136  |tcc|gtt|agC|TCC|TCt|tat|tta|gct|tgg|tat|cag|caa|aag|ccg|ggt|             BseRI...   FR2---------------------------  CDR2-----------------------   61  62  63  64  65  66  67  68  69  70  71  72  73  74  75    Q   A   P  R45  L   L   I   Y  G50  A   S   S   R   A  T56 181  |caa|gct|CCG|CGG|ctg|ttg|atc|tat|ggt|gcc|tct|agt|cgt|gct|act|           SacII..   FR3-------------------------------------------------------   76  77  78  79  80  81  82  83  84  85  86  87  88  89  90    G   I   P  D60  R   F   S   G  S65  G   S   G   T   D   F 226  |ggc|atc|cct|gat|cgt|ttc|tct|ggc|tct|ggc|TCC|GGA|acc|gat|ttc|                                           BspEI..   FR3-------------------------------------------------------   91  92  93  94  95  96  97  98  99  100 101 102 103 104 105    T   L   T   I   S   R   L   E   P   E   D   F   A   V   Y 271  |act|ctg|acc|att|tct|CGT|CTC|GAG|ccg|gaa|gat|ttc|gct|gtc|tac|                       BsmBI..                           XhoI...   FR3---- CDR3------------------------------ FR4-----------  106 107 108 109 110 111 112 113 114 115 116 117 118 119 120    Y   C  Q89  Q   Y   G   S   S  P95  L   T   F   G   G   G 316  |tat|tgt|caa|cag|tat|ggt|tct|agt|ccg|ctg|act|ttc|ggt|ggc|GGT|                                                           KpnI...  FR4--------------------  121 122 123 124 125 126    T   K   V   E   I   K 361  |ACC|aaa|gtc|gaa|atc|aag KpnI.     Ckappa-----------------------------------------------------     R   G   T   V   A   A   P   S   V   F   I   F   P   P   S 379    cgt gga act gtg gCT GCA Cca tct GTC TTC atc ttc ccg cca tct                    BsgI....        BbsI...     D   E   Q   L   K   S   G   T   A   S   V   V   C   L   L 424    gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg     N   N   F   Y   P   R   E   A   K   V   Q   W   K   V   D 469    aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat     N   A   L   Q   S   G   N   S   Q   E   S   V   T   E   Q 514    aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag     D   S   K   D   S   T   Y   S   L   S   S   T   L   T   L 559    gac agc aag gac agc acc tac agc ctc agc agc acc ctg act ctg     S   K   A   D   Y   E   K   H   K   V   Y   A   C   E   V 604    tcc aaa gca gac tac gag aaa cac aaa GTC TAC gcc tgc gaa gtc     T   H   Q   G   L   S   S   P   V   T   K   S   F   N   R 649    acc cat cAG GGC CTg agt tCA CCG GTG aca aag agc ttc aac agg            AlwNI......      SgrAI.....             EcoO109I.(2/2)   AgeI....     G   E   C   .   . 694    gga gag tgt taa taa 709                       GG CGCGCCaatt                       AscI.....                        BssHII.

TABLE 70 Tally of mutations in CDRs of A27 Abs CDR1 (‘RASQSVSSSYLA’ disclosed as SEQ ID NO: 925) R24 1, 3G, 1T, 151-, Fix A25 2, 3T, 152-, Fix S26 3, 1R, 154-, Fix Q27 4, 3E, 1H, 1L, 1P, 4R, 145-, 9% ERYSL S28 5, 1A, 2F, 2G, 11, 2L, 5N, 1P, 1R, 10T, 1V, 1Y, 128-, 9% NTYERL V29 6, 1F, 19I, 6L, 129-, Fix S30 7, 2A, 2D, 8G, 2H, 1I, 11N, 9R, 6T, 4V, 2Y, 108-, 9% DNRTY 530a 8, 1A, 2F, 6G, 1H, 6N, 1P, 10R, 6T, 3Y, 119-, 9% GNRTYD (8% delete 30a) S31 9, 1A, 5D, 3F, 4G, 1H, 2I, 4K, 1L, 31N, 19R, 7T, 7Y, 70-, 9% DFGNRTY Y32 10, 5F, 1K, 14L, 4N, 4Q, 2R, 85, 3V, 1W, 113-, 9% FDLNQRSY L33 11, 16A, 1F, 41, 1N, 1S, 8V, 1Y, 123-, Fix A34 12, 2G, 2L, 1N, 1S, 4V, 128-, 9% SY _ 13, 2A, 1G, _ 14, 1S, _ 15, 1S, _ 16, 1Y, _ 17, 1L, _ 18, 1A, Note: one antibody had an insertion of six AAs in CDR1! Two other Abs had a single insertion. Seventeen Abs have a one AA deletion in CDR1. CDR2 (‘GASSRAT’ disclosed as SEQ ID NO: 926) G50 1, 10A, 11D, 1H, 2R, 2S, 1V, 7Y, 121-, 9% DRSYL A51 2, 7G, 2I, 65, 7T, 2V, 131-, Fix S52 3, 6A, 3F, 1G, 1T, 144-, Fix S53 4, 1A, 1G, 1H, 51, 2K, 16N, 7R, 16T, 106-, 9% NTSYER R54 5, 1A, 1I, 1N, 1S, 3T, 1Y, 147-, Fix A55 6, 2P, 7R, 45, 2V, 140-, Fix T56 7, 10A¹, 1G, 1H, 2P, 4S, 137-, 9% ERSY _ 8, 1A, 6T, Note, there are seven antibodies with an insertion of one AA. CDR3 (showing “_”  means that the Ab has a deletion in CDR3) ('QQYGSSPLT' disclosed as SEQ ID NO: 966) Q89 1, 5H, 1L, 2M, 147-, Fix Q90 2, 1E, 1F, 13H, 2K, 2L, 4R, 1S, 1Y, 130-, Fix Y91 3, 2A, 8F, 2G, 2H, 1L, 1P, 13R, 4S, 122-, 9% FERS G92 4, 10A, 3D, 2H, 1I, 1L, 2N, 6R, 125, 2V, 3Y, 108-, 5_,  9% ADRSTY S93 5, 1A, 2D, 2F, 6G, 2H, 3I, 2K, 2M, 14N, 1P, 1Q, 8R, 17T, 2Y, 86-, 6 ,  9% DFNRTY (8% have 93 deleted) S94 6, 3A, 6F, 1I, 3L, 3P, 2R, 2T, 11W, 117-, 7_,  9% WERYS P95 7, 2A, 1E, 1G, 1K, 3L, 1M, 7R, 3S, 3T, 1V, 108-, 24_,  9% ERYS L96 8, 2A, 2E, 3F, 3G, 1H, 1I, 3K, 7L, 2M, 24P ², 6Q, 28R, 33, 3T, 7V, 2Y, 58-,  9% ERPYS T97 9, 2A, 1F, 2G, 3I, 1K, 2L, 3M, 1N, 1R, 6S, 3V, 2Y, 128-,  Fix _ 10, 1A, 1S, 34-, _ 11, 1S, 7-, _ 12, 1A, There is one Ab with an insertion of 3 AAs. Five have deletions of 4 AAs, 1 has a 3 AA deletion, 1 has a 2 AA deletion, and 17 have a one AA deletion.

TABLE 71 Allowed diversity in CDR1, 2, and 3 of A27::JK4. (‘RASQSVSSSYLA’ disclosed as SEQ ID NO: 925; ‘GASSRAT’ disclosed as SEQ ID NO: 926; ‘QQYGSSPLT’ disclosed as SEQ ID NO: 966) Position parental allowed CDR1 42(24) R fixed 43(25) A fixed 44(26) S fixed 45(27) 4 ERYSL 55% Q 9% other 46(28) S NTYERL 46% S 9% other 47(29) V fixed 48(30) S DNRTY 55% S 9% other 49(30a) S GNRTYD 46% S 9% other 8% have 30a deleted 50(31) S DFGNRTY 44% S 8% other 51(32) Y FDLNQRSY 44% Y 7% other 52(33) L fixed 53(34) A SY 70% A 15% other CDR2 69(50) G DRSYL 55% G 9% other 70(51) A Fixed 71(52) S Fixed 72(53) S NTSYER 52% S 8% other 73(54) R Fixed 74(55) A Fixed 75(56) T ERSY 64% T 9% other CDR3 108(89) Q fixed 109(90) Q fixed 110(91) Y FERS 64% Y 9% other 111(92) G ADRSTY 52% G 8% other 112(93) S DFNRTY 52% S 8% other 113(94) S WERYS 55% S 9% other 114(95) P ERYS 64% P 9% other 8% have P95 deleted 115(96) L ERPYS 55% L 9% other 116(97) T fixed

The parental sequence appears at 5.32 E−5 or 1 in 1.88 E 4. ¹ Seven of these come from the insertions.² Some of these appear because of insertions.

Sequences with a single substitution have a probability between 1.1 E−5 and 7.5 E−6.

Sequences that have none of the parental AAs occurs at 1 in 6.7 E 16.

The allowed diversity is about 2.35 E 12.

TABLE 75 Frequencies of amino acids in HC CDR3s. Rel AA Number % Rel up down Y 3428 15.64 50.41 1.00 G 3244 14.80 47.71 0.95 D 2622 11.96 38.56 0.76 S 1777 8.11 26.13 0.52 R 1337 6.10 19.66 0.39 F 1328 6.06 19.53 0.39 A 1213 5.53 17.84 0.35 V 1141 5.20 16.78 0.33 L 816 3.72 12.00 0.24 I 745 3.40 10.96 0.22 P 726 3.31 10.68 0.21 T 586 2.67 8.62 0.17 W 566 2.58 8.32 0.17 M 560 2.55 8.24 0.16 N 462 2.11 6.79 0.13 E 363 1.66 5.34 0.11 K 355 1.62 5.22 0.10 H 327 1.49 4.81 0.10 Q 259 1.18 3.81 0.08 C 68 0.31 1.00 0.02 Total 21923

TABLE 76 Length distribution of HC CDR3 Number of Length Antibodies Sum Median 1 0 2 0 3 2 2 4 21 23 5 16 39 6 100 139 7 36 175 8 78 253 9 155 408 10 153 561 11 134 695 11.12 12 123 818 13 133 951 14 92 1043 15 87 1130 16 71 1201 17 59 1260 18 41 1301 19 40 1341 20 22 1363 21 21 1384 22 15 1399 23 7 1406 24 7 1413 25 1 1414 26 1 1415 27 3 1418 28 0 1418 29 0 1418 30 1 1419 1419 709.5

TABLE 77 Utilization of D-segments (cut off at 0.70 match) SEQ ID NO: 3-22.2 38 YYYDSSGYYY 88 4-17.2 27 DYGDY 195 3-3.2 25 YYDFWSGYYT 177 6-19.1 25 GYSSGWY 218 7-27.1 19 LTG 221 5-5.3 18 GYSYGY 786 6-13.1 18 GYSSSWY 215 5-12.3 13 GYSGYDY 205 6-13.2 10 GIAAAG 216 1-26.3 9 YSGSYY 284 2-15.2 9 GYCSGGSCYS 136 4-4.3 9 TTVT 190 3-10.2 8 YYYGSGSYYN 81 1-1.3 7 YNWND 262 4-4.2 7 DYSNY 754 2-2.2 6 GYCSSTSCYT 70 3-16.2 6 YYDYVWGSYRYT 104 6-6.1 6 EYSSSS 212 6-19.2 6 GIAVAG 219 3-9.1 5 VLRYFDWLL@ 179 4-23.2 5 DYGGNS 198 6-6.2 5 SIAAR 213 1-7.3 4 YNWNY 270 2-2.3 4 DIVVVPAA I 168 4-23.3 4 TTVVT 199 1-7.1 3 GITGT 268 1-26.1 3 GIVGAT 282 7-27.3 3 NWG 223 3-10.1 2 VLLWFGELL@ 182 3-10.2 2 ITMVRGVII 183 5-5.1 2 VDTAMV 200 5-5.2 2 WIQLWL 201 5-12.1 2 VDIVATI 203 5-24.3 2 RDGYNY 211 1-1.1 1 GTTGT 260 2-21.3 1 HIVVVTA I 175 3-3.3 1 ITIFGVVII 178 5-24.2 1 *RWLQL 210 6-6.3 1 V*QLV 214 6-19.3 1 V*QWLV 220

TABLE 78 D segment utilization (0.667 cutoff) Name Number Sequence SEQ ID NO: % None 935 0.517 7-27.1 158 LTG 221 0.087 7-27.3 98 NWG 223 0.054 5-5.3 72 GYSYGY 786 0.040 1-26.3 67 YSGSYY 166 0.037 3-22.2 46 YYYDSSGYYY 187 0.025 4-17.2 38 DYGDY 195 0.021 3-3.2 37 YYDFWSGYYT 177 0.020 7-27.2 37 @LG 222 0.020 6-19.1 33 GYSSGWY 218 0.018 6-13.2 31 GIAAAG 860 0.017 6-13.1 22 GYSSSWY 215 0.012 6-6.1 18 EYSSSS 847 0.010 6-19.2 18 GIAVAG 879 0.010 4-23.2 17 DYGGNS 198 0.009 5-12.3 17 GYSGYDY 205 0.009 5-24.3 14 RDGYNY 211 0.008 2-15.2 13 GYCSGGSCYS 136 0.007 1-26.1 11 GIVGAT 164 0.006 4-4.3 11 TTVT 190 0.006 1-1.3 9 YNWND 262 0.005 2-2.2 9 GYCSSTSCYT 70 0.005 3-16.2 9 YYDYVWGSYRYT 104 0.005 2-2.3 8 DIVVVPAAI 168 0.004 3-10.2 8 YYYGSGSYYN 81 0.004 4-4.2 8 DYSNY 192 0.004 1-7.3 7 YNWNY 270 0.004 3-3.3 6 ITIFGVVII 178 0.003 6-6.2 6 SIAAR 213 0.003 3-9.1 5 VLRYFDWLL@ 179 0.003 3-10.2 5 ITMVRGVII 183 0.003 6-19.3 5 V*QWLV 220 0.003 1-7.1 4 GITGT 268 0.002 4-23.3 4 TTVVT 768 0.002 1-1.1 3 GTTGT 156 0.002 5-5.1 3 VDTAMV 200 0.002 5-24.2 3 *RWLQL 210 0.002 3-10.1 2 VLLWFGELL@ 182 0.001 5-5.2 2 WIQLWL 201 0.001 5-12.1 2 VDIVATI 203 0.001 1-26.2 1 V*WELL 165 0.001 2-21.2 1 AYCGGDCYS 174 0.001 2-21.3 1 HIVVVTAI 175 0.001 3-3.1 1 VLRFLEWLLY 176 0.001 3-16.2 1 IMITFGGVIVI 185 0.001 6-6.3 1 V*QLV 214 0.001 6-13.3 1 V*QQLV 217 0.001

TABLE 78 Utilization of JH segments JH1 17 JH2 31 JH3 452 JH4 636 JH5 32 JH6 251

Example 6: Wobbled DNA for HC CDR3 16d

Table 400 shows a segment of DNA from an XbaI site in FR3 to a BstEII site in FR4. The HC CDR3 consists of SYSY::D2-2(2)::QH (‘SYSY’ disclosed as SEQ ID NO: 947) followed by the FR4 region of JH1. The QH is found in the germline of JH1. In V-D-J joining, immune cells often edit the ends of V, D, and J. Thus the construction corresponds to what is very possible in actual immunoglobulin gene construction and maturation. By wobbling the synthesis, we obtain a large collection of genes that resemble what would come from joining 3-23 to either a D region or to a little edited JH1 followed by some mutations. In library 16d, there are two cysteines that presumably form a disulfide, these are not wobbled.

Table 500 shows the expected distribution of amino-acid types at each position in the 16d library. The wobble doping was set at 73:9:9:9. The most likely sequence is the one shown in Table 21 and should be present at a frequency of 4.8 E-5. Only 55% of the sequences are stop free and 74% are free of ochre or opel. If the library is expressed in supE cells, this is the important number. It would be valuable to remove the sequences with stop codons as discussed elsewhere herein. One can see that those positions that start as S are predicted to have S 54% of the time and Y 5.4% while those that start as Y have Y 44% of the time and S 7.2%. At each position there are 7-9 AA types that appear at >1%. There are 14 variegated positions. The sequences that will be most effectively sampled number about 8¹⁴=4.3 E 12.

TABLE 400 Cassette for display of wobbled HC CDR3 16d --------FR3--------------------------------------------------  68  69  70  71  72  73  74  75  76  77  78  79  80  81  82   T   I   S   R   D   N   S   K   N   T   L   Y   L   Q   M 1216 |act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|         | XbaI  | ---FR3----------------------------------------------------->| 82a 82b 82c  83  84  85  86  87  88  89  90  91  92  93  94   N   S   L   R   A   E   D   T   A   V   Y   Y   C   A   K 1261 |aac|agC|TTA|AGg|gct|gag|gac|act|gca|gtc|tac|tat|tgc|gct|aaa|            |AflII |  e = 0.73 A + 0.09 C + 0.09 G + 0.09 T q = 0.09 A + 0.73 C + 0.09 G + 0.09 T j = 0.09 A + 0.09 C + 0.73 G + 0.09 T z = 0.09 A + 0.09 C + 0.09 G + 0.73 T The values 0.73 and 0.09 are picked so that 0.73 + 3 * 0.09 = 1.0 Other ratios could be used.                                 102 102 102 102 102 102 102 102 95  96  97  98  99  100 101 102  a   b   c   d   e   f   g   h  S   Y   S   Y   G   Y   c   S   S   T   S   c   Y   T   Q   H zqz zez zqz zez jjz zez TGT zqz zqz eqz zqz TGT zez eqz qej qez  --------------FR4------------------------->|  103 104 105 106 107 108 109 110 111 112 113   W   G   Q   G   T   L   V   T   V   S   S (SEQ ID NO: 968) |TGg|ggt|caa|ggt|act|ttG|GTC|ACC|gtc|tct|agt (SEQ ID NO: 967)                        | BstEII |

TABLE 500 Expected distribution of AA types in wobbled HC CDR3 16d “.” = TGA or TAA; “b”= TAG Amino acid disclosed as SEQ ID NO: 970 DNA sequence disclosed as SEQ ID NO: 969  S   Y   S   Y   G   Y   c   S   S   T   S   c   Y   T   Q   H zqz zez zqz zez jjz zez tgt zqz zqz eqz zqz tgt zez eqz qej qez Nominal base purity = 0.7300 others = 0.0900 s(zqz) y(zez) s(zqz) y(zez) g(jjz) y(zez) C(TGT) s (zqz) s(zqz)  1 s 5.4-01 y 4.4-01 s 5.4-01 y 4.4-01 g 5.3-01 y 4.4-01  c 1.000 s 5.4-01 s 5.4-01  2 p 6.6-02 s 7.2-02 p 6.6-02 s 7.2-02 r 7.8-02 s 7.2-02 p 6.6-02 p 6.6-02  3 a 6.6-02 f 5.4-02 a 6.6-02 f 5.4-02 a 6.6-02 f 5.4-02 a 6.6-02 a 6.6-02  4 t 6.6-02 h 5.4-02 t 6.6-02 h 5.4-02 v 6.6-02 h 5.4-02 t 6.6-02 t 6.6-02  5 f 5.4-02 n 5.4-02 f 5.4-02 n 5.4-02 s 6.2-02 n 5.4-02 f 5.4-02 f 5.4-02  6 c 5.4-02 c 5.4-02 c 5.4-02 c 5.4-02 c 5.4-02 c 5.4-02 c 5.4-02 c 5.4-02  7 y 5.4-02 d 5.4-02 y 5.4-02 d 5.4-02 d 5.4-02 d 5.4-02 y 5.4-02 y 5.4-02  8 l 2.0-02 . 5.4-02 l 2.0-02 . 5.4-02 e 1.2-02 . 5.4-02 l 2.0-02 l 2.0-02  9 . 1.2-02 b 4.8-02 . 1.2-02 b 4.8-02 l 9.6-03 b 4.8-02 . 1.2-02 . 1.2-02 10 r 9.6-03 l 2.0-02 r 9.6-03 l 2.0-02 t 8.1-03 l 2.0-02 r 9.6-03 r 9.6-03 11 g 8.1-03 k 1.2-02 g 8.1-03 k 1.2-02 p 8.1-03 k 1.2-02 g 8.1-03 g 8.1-03 12 v 8.1-03 q 1.2-02 v 8.1-03 q 1.2-02 i 7.4-03 q 1.2-02 v 8.1-03 v 8.1-03 13 i 7.4-03 e 1.2-02 i 7.4-03 e 1.2-02 . 6.6-03 e 1.2-02 i 7.4-03 i 7.4-03 14 h 6.6-03 r 9.6-03 h 6.6-03 r 9.6-03 f 6.6-03 r 9.6-03 h 6.6-03 h 6.6-03 15 n 6.6-03 t 8.1-03 n 6.6-03 t 8.1-03 h 6.6-03 t 8.1-03 n 6.6-03 n 6.6-03 16 d 6.6-03 v 8.1-03 d 6.6-03 v 8.1-03 y 6.6-03 v 8.1-03 d 6.6-03 d 6.6-03 17 w 5.9-03 a 8.1-03 w 5.9-03 a 8.1-03 n 6.6-03 a 8.1-03 w 5.9-03 w 5.9-03 18 b 5.9-03 g 8.1-03 b 5.9-03 g 8.1-03 w 5.9-03 g 8.1-03 b 5.9-03 b 5.9-03 19 q 1.5-03 p 8.1-03 q 1.5-03 p 8.1-03 q 1.5-03 p 8.1-03 q 1.5-03 q 1.5-03 20 k 1.5-03 i 7.4-03 k 1.5-03 i 7.4-03 k 1.5-03 i 7.4-03 k 1.5-03 k 1.5-03 21 e 1.5-03 w 5.9-03 e 1.5-03 w 5.9-03 m 7.3-04 w 5.9-03 e 1.5-03 e 1.5-03 22 m 7.3-04 m 7.3-04 m 7.3-04 m 7.3-04 b 7.3-04 m 7.3-04 m 7.3-04 m 7.3-04 t(eqz) s(zqz) C(TGT) y(zez) t(eqz) q(qej) h(qez)  1 t 5.3-01 s 5.4-01 c 1.000 y 4.4-01 t 5.3-01 q 4.4-01 h 4.4-01  2 s 1.2-01 p 6.6-02 s 7.2-02 s 1.2-01 h 9.6-02 q 9.6-02  3 a 6.6-02 a 6.6-02 f 5.4-02 a 6.6-02 l 7.2-02 l 6.7-02  4 p 6.6-02 t 6.6-02 h 5.4-02 p 6.6-02 r 7.2-02 r 6.7-02  5 i 6.0-02 f 5.4-02 n 5.4-02 i 6.0-02 p 6.6-02 p 6.6-02  6 n 5.4-02 c 5.4-02 c 5.4-02 n 5.4-02 e 5.4-02 n 5.4-02  7 r 2.0-02 y 5.4-02 d 5.4-02 r 2.0-02 k 5.4-02 d 5.4-02  8 k 1.2-02 l 2.0-02 . 5.4-02 k 1.2-02 b 4.8-02 y 5.4-02  9 l 9.6-03 . 1.2-02 b 4.8-02 l 9.6-03 d 1.2-02 s 1.5-02 10 g 8.1-03 r 9.6-03 l 2.0-02 g 8.1-03 y 1.2-02 k 1.2-02 11 v 8.1-03 g 8.1-03 k 1.2-02 v 8.1-03 n 1.2-02 e 1.2-02 12 f 6.6-03 v 8.1-03 q 1.2-02 f 6.6-03 s 9.6-03 g 8.1-03 13 c 6.6-03 i 7.4-03 e 1.2-02 c 6.6-03 t 8.1-03 t 8.1-03 14 h 6.6-03 h 6.6-03 r 9.6-03 h 6.6-03 v 8.1-03 v 8.1-03 15 d 6.6-03 n 6.6-03 t 8.1-03 d 6.6-03 a 8.1-03 a 8.1-03 16 y 6.6-03 d 6.6-03 v 8.1-03 y 6.6-03 g 8.1-03 i 7.4-03 17 m 5.9-03 w 5.9-03 a 8.1-03 m 5.9-03 . 6.6-03 . 6.6-03 18 q 1.5-03 b 5.9-03 g 8.1-03 q 1.5-03 w 5.9-03 c 6.6-03 19 e 1.5-03 q 1.5-03 p 8.1-03 e 1.5-03 m 5.9-03 f 6.6-03 20 . 1.5-03 k 1.5-03 i 7.4-03 . 1.5-03 i 2.2-03 b 5.9-03 21 w 7.3-04 e 1.5-03 w 5.9-03 w 7.3-04 f 1.5-03 w 7.3-04 22 b 7.3-04 m 7.3-04 m 7.3-04 b 7.3-04 c 1.5-03 m 7.3-04 Most likely sequence has frequency = 4.8E−05 Fraction stop-free = 5.5E−01 Fraction (TAA&TGA)-free = 7.4E−01 F % F % F % D1 1-1  0.42  0.14  2.90 1-7  0.42  0.28  1.24  1-20  0.00  0.00  0.00  1-26  0.00  0.97  1.80 D2 2-2  0.55  4.30  1.21 2-8  0.00  0.67  0.41  2-15  0.28  4.03  0.94  2-21  0.00  2.22  0.94 D3 3-3  0.94  4.44  3.70 3-9  0.67  1.82  0.00  3-10  0.67  5.78  1.55  3-16  1.08  2.49  0.67  3-22  0.14  7.87  0.81 D4 4-4  0.28  0.69  0.28  4-11  0.00  0.00  0.00  4-17  0.00  4.03  2.76  4-23  0.14  1.41  0.54 D5 5-5  1.34  0.40  4.30  5-12  1.08  0.00  1.95  5-18  0.00  0.00  0.00  5-24  0.67  1.55  1.82 D6 6-6  1.21  1.55  0.13  6-13  4.84  2.62  0.27  6-19  6.66  1.95  0.54 D7  7-27  0.27  0.13  0.27 Total fractional % 21.65 49.34 29.01

TABLE 800 !LC K1(O12)::JK1 !Amino acid disclosed as SEQ ID NO: 972 DNA sequence disclosed as SEQ ID NO: 971 ! ..Leader seq. ->|-------- FR1 -----------------------------> !                           1   2   3   4   5   6   7   8   9  10  11 !   G   V   H   S   A   Q   D   I   Q   M   T   Q   S   P   S   S   L   1 |ggT|GTA|CAc|aGT|GCT|Cag|gat|att|cag|atg|act|caa|tct|ccC|TCG|AGt|ctg| !    BsrGI...   ApaLI...                                 XhoI.... ! -------- FR1 ---------------------------------->|--- CDR1 -> !  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26 !   S   A   S   V   G   D   R   V   T   I   T   C   R   A   S  46 |tct|gct|tct|gtc|gGC|GAT|CGC|gtt|act|att|act|tgt|cgt|gct|tcc| !                   SgfI...... ! ---- CDR1 -------------------->|---- FR2 -----------------> !  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41 !   Q   S   I   S   S   Y   L   N   W   Y   Q   Q   K   P   G  91 |cag|tcc|att|tct|agc|tat|ctg|aat|tGG|TAC|Cag|caa|aag|ccg|ggt| !                                   KpnI.... ! ! ------ FR2 ------------------->|-- CDR2 ------------------> !  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56 !   K   A   P   K   L   L   I   Y   A   A   S   S   L   Q   S 136 |aag|gct|ccg|aaa|ctg|tta|atc|tat|gcc|gct|tct|agt|ctg|cag|tct| ! ---------- FR3 -------------------------------------------> !  57  58  59  60  61  62  63  64  65  66  67  68  69  70  71 !   G   V   P   S   R   F   S   G   S   G   S   G   T   D   F 181 |ggt|gtt|ccg|TCT|AGA|ttc|tct|ggc|tct|ggt|tct|ggt|act|gat|ttt| !              XbaI... ! ---------- FR3 -------------------------------------------> !  72  73  74  75  76  77  78  79  80  81  82  83  84  85  86 !   T   L   T   I   S   S   L   Q   P   E   D   F   A   T   Y 226 |act|ctg|act|att|tcc|tct|ctg|caa|ccg|gag|gac|ttt|gct|acc|tat| ! - FR3>|---- CDR3 ------------------------>|--- FR4 ------> !  87  88  89  90  91  92  93  94  95  96  97  98  99  100 101 !   Y   C   Q   Q   S   Y   S   T   P   W   T   F   G   Q   G 271 |tac|tgc|caa|cag|tct|tat|agt|act|ccg|tgg|act|ttc|ggt|caa|ggc| ! ---- FR4 -------------->|---- Ckappa-----------------------> !  102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 !   T   K   V   E   I   K   R   T   V   A   A   P   S   V   F 316 |act|aaa|gtt|gag|att|aag|CGT|ACG|gtg|gct|gct|ccg|tct|gtc|ttc| !                          BsiWI..

TABLE 900 CDR1 diversity (SEQ ID NO: 973) Diversity Position 24 25 26 27 28 29 30 31 32 33 34 O12 R A S Q S I S S Y L N diversity 2 2 1 1 3 1 2 2 4 1 3 576 allowed Q M D R N D A G W G A

TABLE 1000 Big CDR1 diversity Diversity Position 24 25 26 27 28 29 30 31 32 33 34 O12 R A S Q S I S S Y L N diversity 3 2 4 1 5 1 4 5 5 1 6 72000 allowed Q M E D R N D A E R G E E W G Y R Y R A D Y Y R R Y

TABLE 1100 CDR2 diversity POSITION 50 51 52 53 54 55 56 Diversity O12 A A S S L Q S diversity 2 1 1 3 1 2 2 24 allowed D N E T T

TABLE 1200 Big CDR2 diversity POSITION 50 51 52 53 54 55 56 Diversity O12 A A S S L Q S diversity 4 1 4 6 1 4 5 1920 allowed D E N E T R R T R Y Y Y E Y R R E Y

TABLE 1300 CDR3 diversity Position 93 94 95 96 97 98 99 100 101 div. tot. O12 Q Q S Y S S P W T diversity 2 2 6 3 3 5 2 1 1 2160 allowed L K Y D N T S H N Y L F Y A F D

TABLE 1400 Big CDR3 diversity Position 93 94 95 96 97 98 99 100 101 div. tot. O12 Q Q S Y S S P W T diversity 6 1 7 7 6 5 2 6 1 105840 allowed L Y D N T S F E H N Y L Y R F R D Y H Y A A R F L A D L A E I R S R

Example 7: Further Examples of Synthetic HC CDR3s

A collection of 22,063 Fabs with distinct CDR3 which had been selected from the FAB-310 or FAB-410 library and which were ELISA positive for at least one antigen were examined. The utilization of JH chains is shown in Table 1001; the FR4 part of each JH is shown bold. Table 1010 shows the utilization of amino acids in the HC CDR3s. Table 1020 shows the length distribution of CDR3. The median length is 11.5.

Table 1030 shows the utilization of D segments in the CDR3s. A D segment was identified is 70% of the amino acids matched; there were 5,654 cases (25.6%). The most used Ds were 3-3.2 (743, sequence: YYDFWSGYYT (SEQ ID NO: 177)), 3-22.2 (617, sequence: YYYDSSGYYY (SEQ ID NO: 88)), 6-19.1 (441, sequence: GYSSGWY (SEQ ID NO: 218)), 6-13.1 (399, sequence: GYSSSWY (SEQ ID NO: 215)), and 4-17.2 (392, sequence: DYGDY (SEQ ID NO: 760)). Of the Ds containing paired Cys residues, 2-15.2 (sequence: GYCSGGSCYS (SEQ ID NO: 136)) was the most used; there were 139 examples which is 0.6% of the collection.

When V or V::D is joined to J, there is often editing of the 3′ end of V or V::D and the 5′ end of J. Inspection of many CDR3-FR4 sequences shows that there is often a portion of JH making up part of CDR3. Often there are mutations in the CDR3 residues corresponding to JH residues 1-9. Herein the portion of CDR3 that is thought to derive from JH is called the “J stump”. The JH used in a heavy chain is determined by comparing each of the residues of the six JH chains from position 6 to 20 to fusion of the last four amino acids of CDR3 to FR4. The JH that has the fewest mismatches is selected. The CDR3 sequence is examined for a J stump by working backward in the selected JH from position 9 toward the first position of the selected JH comparing to CDR3 until the search is terminated by a) the end of JH, b) the end of CDR3, or c) two consecutive mismatches. If one of the chains ends and the last compared position is a match, then it is included in J stump. If not, it is not. Table 1070 shows several examples. The CDR is written above, the JH is below, and the J stump is underlined. In 1070 A, we start at 9, V matches V, and we continue to position 6 with matches. The search stops at 4 because of the double mismatch. GMDV (SEQ ID NO: 974) goes into the J stump pile and GL goes into the “Leadin” pile. In 1070 B, the search ends with the end of JH6. The underscored residues go into the J stump pile and EPIWG (SEQ ID NO: 975) goes into the Leadin pile. In 1070 E, the search terminates because of the end of JH4, but the final residue tested (D in the CDR vs Y in JH4) is a mismatch and so the J stump is FDS and DSGVVAAAD (SEQ ID NO: 976) goes into the Leadin pile.

Table 1015 shows the amino-acid distribution of CDR3s that have no D segments from which the J stump has been removed. Note that the frequency of Tyr is much lower than when the whole CDR3s were compiled. This indicates that Tyr comes into CDR3s to a large extent through incorporation of D segments and J stumps. These Tyrs are not randomly inserted, but occur in a sequence that has been selected throughout mammalian evolution. It is a feature of the present invention that high levels (more than 20%) of Tyr should be inserted into libraries through the incorporation of Ds and J stumps that contain Tyr. At leadin or DJ filler positions, Tyr is allowed, but at no more than 20%.

TABLE 1070 Examples of assignment of J stump A)  6    GLGMDV                (SEQ ID NO: 977) JH6 YYYYYGMDVWGQGTTVTVSS     (SEQ ID NO: 3) 123456789 B) 13 EPIWGYYYYGMDV            (SEQ ID NO: 978) JH6     YYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 3) C)  9 DFFTSYFDY                (SEQ ID NO: 979) JH4 -----YFDYWGQGTLVTVSS     (SEQ ID NO: 1) D) 12 DRGVSLLGAFDI             (SEQ ID NO: 980) JH3    -----AFDIWGQGTMVTVSS  (SEQ ID NO: 2) E) 12 DSGVVAAADFDS             (SEQ ID NO: 981) JH4    -----YFDYWGQGTLVTVSS  (SEQ ID NO: 1)         6789

Table 1082 shows the distribution of amino-acid usage in the J stumps of each JH. Since the most common JHs are JH3, JH4, and JH6, these are the preferred JHs on which to build libraries. Table 1082 shows that most examples of JH3 retain the tetrapeptide sequence AFDI (SEQ ID NO: 986) in CDR3. With JH4, a majority retain DY and a large fraction retain the sequence FDY in CDR3. With JH6, a large majority retain the sequence DV, a majority retain the sequence MDV, and a substantial fraction retain the sequence GMDV (SEQ ID NO: 974). A non-negligible fraction retain the sequence YGMDV (SEQ ID NO: 982), YYGMDV (SEQ ID NO: 983), or YYYGMDV (SEQ ID NO: 984).

Included in libraries of the present invention are libraries such as 5.001 (Table 1097). Library 5.001 contains LC and HC CDR1-2 as described elsewhere in the present application. The library contains a HC VH (such as 3-23) followed by 6, 7, or 8 amino acids allowing [GSRDLY] in proportion shown in Table 1097. In the J stump, the parental amino acid is present at 3, 4, 5, 6, 7, 8, 10 times as likely as “other” amino-acid types. The “other” amino-acid types comprise Y, S, D, R, G. Thus at A6, we allow 7/12 A, plus 1/12 each of Y, S, D, R, and G. At F7, we allow 7/12 F plus 1/12 each of Y, S, D, R, and G. At D8, we allow 7/11 D plus 1/11 of Y, S, R, and G. At 19, we allow 7/12 I plus 1/12 Y, S, R, D, G. The parental amino acid could be 5, 6, 7, 8, 10 time more likely than the other amino-acid types.

Included in the libraries of the present invention is library 5.002 in Table 1097. This library comprises CDR3 of length 13, 14, and 15 and no D segment. There are 6, 7, or 8 leadin residues allowing G, S, R, D, L, or Yin the ratios 1:0.57:0.46:0.42:0.36:0.35 or reasonable approximation thereto. The CDR3 is completed with a portion of JH6: YYYGMDV (SEQ ID NO: 984). The DNA that encodes the parental sequence YYYGMDV (SEQ ID NO: 984) is synthesized with the parental amino acid at 5, 6, 7, 8, or 10 times more likely than the others.

Included in the library of the present invention is library 5.003 in Table 1097. FR3 is followed by 4, 5, or 6 leadin residues allowing G, S, R, D, L, Y in the ratio 1.0:0.57:0.46:0.42:0.36:0.35. Next comes D segment 3-3.2; the DNA that encodes this region favors the parental amino acid by 5-fold and allows as other amino acids Y, G, D, R, S. There is no DJ filler and the final four amino acids come from the J stump of JH3. The DNA encoding the J stump are synthesized with the parental amino acid 5-fold more likely than the others: YSGRD.

Library 5.004 in Table 1097 is a part of the present invention. There are 2, 3, or 4 leadin residues allowing GSRDLY in the ratios shown. The DNA encoding the sequence GYSSGWY (SEQ ID NO: 218) is synthesized so that the parental amino acid is 6-X as likely as the others, two DJ-filler residues are allowed with GSRDLY allowed in the ratios 1.0:0.57:0.46:0.42:0.36:0.35. The DNA to encode AFDI (SEQ ID NO: 986) is synthesized with the parental amino acid 6-x as likely as the others.

Library 5.005 is part of the present invention. Library 5.005 comprises members with CDR3 lengths 11-14. After FR3, there are 0, 1, or 2 leadin residues allowing GSRDLY in the ratios shown followed by DNA that encodes the parental sequence GYSSGWY (SEQ ID NO: 218) with variability that allows YGSRD such that the parental amino acid is 6-X as likely as the other allowed types. Following the D region there is zero or one DJ filler residues allowing GSRDLY in the ratios shown. Finally is JH3 with variability in the J stump (sequence: YFDY (SEQ ID NO: 985)) which allows YGSRD with the parental amino acid 6-X as likely as the other allowed types.

Library 5.006 in Table 1097 is part of the present invention. The CDR3 may be of length 19-25. There are zero to three leadin residues allowing GSRDLY in the ratios shown. Following the leadin is the D region 2-2.2. The DNA encoding 2-2.2 is synthesized so that the parental amino acid is 6-X as likely as the others (viz. YGSRD) except that the two Cys residues are fixed. Following 2-2.2 are zero to three DJ filler residues allowing GSRDLY in the ratios shown. The DNA that encodes the first nine residues of JH6 allows the parental amino acid plus YSGDR with the parental type being 6× more likely than the others.

TABLE 1001 Utilization of JHs          11111111112 SEQ ID JH Number % 12345678901234567890 NO: JH1  1356 6.15 ---AEYFQHWGQGTLVTVSS 66 JH2  1720 7.80 ---YWYFDLWGRGTLVTVSS 67 JH3  5601 25.39 -----AFDIWGQGTMVTVSS  2 JH4  7658 34.71 -----YFDYWGQGTLVTVSS  1 JH5  1062 4.81 ----NWFDPWGQGTLVTVSS 68 JH6  4666 21.15 YYYYYGMDVWGQGTTVTVSS  3 Total 22063

TABLE 1010 Utilization of Amino acids in HC CDR3 Rel Rel AA Number % up dwn Y  42863 15.47 35.87 1.00 G  37512 13.54 31.39 0.88 D  34051 12.29 28.49 0.79 S  23068  8.33 19.30 0.54 F  17813  6.43 14.91 0.42 A  15150  5.47 12.68 0.35 R  14090  5.09 11.79 0.33 V  13834  4.99 11.58 0.32 L  12351  4.46 10.34 0.29 I  10014  3.61  8.38 0.23 P   9514  3.43  7.96 0.22 W   9340  3.37  7.82 0.22 T   7544  2.72  6.31 0.18 M   6093  2.20  5.10 0.14 E   6042  2.18  5.06 0.14 N   5901  2.13  4.94 0.14 H   4403  1.59  3.68 0.10 K   3147  1.14  2.63 0.07 Q   3097  1.12  2.59 0.07 C   1195  0.43  1.00 0.03 277022

TABLE 1015 Frequency of amino acids in Leadin of CDR3s lacking D regions AA Number percent rel up rel dn G 23134 18.24 46.45 1.000 S 13555 10.69 27.22 0.586 R 10562 8.33 21.21 0.457 D 9704 7.65 19.49 0.419 L 8255 6.51 16.58 0.357 Y 8099 6.39 16.26 0.350 A 7188 5.67 14.43 0.311 V 6599 5.20 13.25 0.285 P 5768 4.55 11.58 0.249 W 4804 3.79 9.65 0.208 T 4769 3.76 9.58 0.206 E 4497 3.55 9.03 0.194 N 3733 2.94 7.50 0.161 F 3616 2.85 7.26 0.156 I 3464 2.73 6.96 0.150 H 2787 2.20 5.60 0.120 K 2460 1.94 4.94 0.106 Q 2124 1.67 4.27 0.092 M 1225 0.97 2.46 0.053 C 498 0.39 1.00 0.022 126841

TABLE 1020 Lengths of HC CDR3s Length Number % 1 0 0.00 2 6 0.03 3 36 0.16 4 153 0.69 5 121 0.55 6 669 3.03 7 756 3.43 8 1066 4.83 9 2227 10.09 10 2701 12.24 11 2240 10.15 12 2071 9.39 13 2006 9.09 14 1594 7.22 15 1396 6.33 16 1254 5.68 17 1102 4.99 18 783 3.55 19 588 2.67 20 474 2.15 21 285 1.29 22 237 1.07 23 133 0.60 24 81 0.37 25 32 0.15 26 25 0.11 27 11 0.05 28 6 0.03 29 2 0.01 30 3 0.01 31 2 0.01 32 1 0.00 33 1 0.00 34 0 0.00 35 0 0.00 36 1 0.00 22063

TABLE 1030 Utilization of D segments. Id Number Sequence SEQ ID NO: 1-1.1 29 GTTGT 156 1-1.2 6 VQLER 157 1-1.3 151 YNWND 158 1-7.1 34 GITGT 159 1-7.2 0 V*LEL 160 1-7.3 65 YNWNY 161 1-20.1 0 GITGT 268 1-20.2 0 V*LER 162 1-20.3 0 YNWND 163 1-26.1 48 GIVGAT 164 1-26.2 3 V*WELL 165 1-26.3 220 YSGSYY 166 2-2.1 0 RIL**YQLLY 177 & 167 2-2.2 102 GYCSSTSCYT  70 2-2.3 37 DIVVVPAAI 168 2-8.1 0 RILY@WCMLY 169 & 392 2-8.2 23 GYCTNGVCYT 115 2-8.3 1 DIVLMVYAI 170 2-15.1 0 RIL*WW*LLL 171 2-15.2 139 GYCSGGSCYS 136 2-15.3 12 DIVVVVAAT 172 2-21.1 0 SILWW$LLF 173 2-21.2 24 AYCGGDCYS 174 2-21.3 6 HIVVVTAI 175 3-3.1 28 VLRFLEWLLY 176 3-3.2 743 YYDFWSGYYT 177 3-3.3 15 ITIFGVVII 178 3-9.1 41 VLRYFDWLL@ 179 3-9.2 8 YYDILTGYYN 180 3-9.3 0 ITIF*LVII 181 & 579 3-10.1 26 VLLWFGELL@ 182 3-10.2 136 YYYGSGSYYN  81 3-10.2 32 ITMVRGVII 183 3-16.1 0 VL$LRLGELSLY 184 3-16.2 109 YYDYVWGSYRYT 104 3-16.2 8 IMITFGGVIVI 185 3-22.1 0 VLL***WLLL 186 3-22.2 617 YYYDSSGYYY 187 3-22.3 2 ITMIVVVIT 188 4-4.1 0 $LQ@L 189 4-4.2 75 DYSNY 192 4-4.3 165 TTVT 190 4-11.1 0 $LQ@L 191 4-11.2 0 DYSNY 192 4-11.3 0 TTVT 193 4-17.1 0 $LR@L 194 4-17.2 392 DYGDY 195 4-17.3 0 TTVT 196 4-23.1 0 $LRW@L 197 4-23.2 60 DYGGNS 198 4-23.3 16 TTVVT 199 5-5.1 25 VDTAMV 200 5-5.2 29 WIQLWL 201 5-5.3 292 GYSYGY 202 5-12.1 13 VDIVATI 203 5-12.2 0 WI*WLRL 204 5-12.3 200 GYSGYDY 205 5-18.1 0 VDTAMV 206 5-18.2 0 WIQLWL 207 5-18.3 0 GYSYGY 208 5-24.1 9 VEMATI 209 5-24.2 21 *RWLQL 210 5-24.3 44 RDGYNY 211 6-6.1 87 EYSSSS 212 6-6.2 122 SIAAR 213 6-6.3 1 V*QLV 214 6-13.1 399 GYSSSWY 215 6-13.2 170 GIAAAG 216 6-13.3 0 V*QQLV 217 6-19.1 441 GYSSGWY 218 6-19.2 104 GIAVAG 219 6-19.3 3 V*QWLV 220 7-27.1 257 LTG 221 7-27.2 0 @LG 222 7-27.3 64 NWG 223 none 16409

TABLE 1040 JH vs Length Length JH1 JH2 JH3 JH4 JH5 JH6  1  0  0  0   0  0  0  2  1  4  0   1  0  0  3  20  2  3   9  0  2  4  75  3  10  45  8  12  5  47  6  10  38  8  12  6 273  14  43  280  26  33  7  88  27 194  337  30  80  8 134  43 243  503  41 102  9 121  70 855  886  61 234 10 116 693 623  979  68 222 11 105  81 675 1003  84 292 12 107  84 552  905 121 302 13  87 274 538  672 113 322 14  48  81 480  532 105 348 15  50  83 372  421  80 390 16  28  54 316  322  87 447 17  27  49 239  334  69 384 18  11  64 174  140  49 345 19  8  28 104  99  41 308 20  4  23  59  56  20 312 21  0  13  40  30  24 178 22  3  14  31  30  13 146 23  1  3  22  12  7  88 24  0  5  9  12  4  51 25  1  0  1   3  1  26 26  0  0  5   5  0  15 27  0  1  2   1  1  6 28  1  0  0   2  0  3 29  0  0  0   0  0  2 30  0  0  0   0  0  3 31  0  0  1   0  0  1 32  0  1  0   0  0  0 33  0  0  0   1  0  0 34  0  0  0   0  0  0 35  0  0  0   0  0  0 36  0  0  0   0  1  0

TABLE 1050 Utilization of amino acids in Leadin with no D segment Rel Rel AA Number % up dn G 23134 18.24 46.45 1.00 S 13555 10.69 27.22 0.59 R 10562 8.33 21.21 0.46 D 9704 7.65 19.49 0.42 L 8255 6.51 16.58 0.36 Y 8099 6.39 16.26 0.35 A 7188 5.67 14.43 0.31 V 6599 5.20 13.25 0.29 P 5768 4.55 11.58 0.25 W 4804 3.79 9.65 0.21 T 4769 3.76 9.58 0.21 E 4497 3.55 9.03 0.19 N 3733 2.94 7.50 0.16 F 3616 2.85 7.26 0.16 I 3464 2.73 6.96 0.15 H 2787 2.20 5.60 0.12 K 2460 1.94 4.94 0.11 Q 2124 1.67 4.27 0.09 M 1225 0.97 2.46 0.05 C 498 0.39 1.00 0.02 126841

TABLE 1080 Dipepides in HC CDR3s, part 1 YY 13565 FG 1073 PL 591 TV 397 FD 11637 RS 1072 TT 589 TP 390 DY 8337 SW 1014 ID 588 NA 389 SG 5979 DW 1003 DD 583 NS 388 GY 5805 LR 990 AS 570 ER 387 YG 5461 DG 989 KG 566 HG 386 DI 5448 PG 976 VD 556 VW 381 AF 4975 LL 974 VP 551 QL 378 DV 4968 AY 962 LT 540 RI 374 GG 4575 DR 923 LF 539 WN 365 SS 4491 VR 882 VL 539 YT 365 MD 4436 YM 877 FY 534 CS 360 GS 4047 AR 872 PD 533 DH 359 GM 3501 VV 869 RV 531 EA 359 YF 3438 YR 865 RF 525 WD 353 YD 3430 VA 857 AL 521 ES 350 RG 3118 RA 844 PS 510 FR 349 SY 2770 SP 820 EY 508 YC 343 GA 2611 GN 812 LW 508 PT 337 YS 2576 HY 809 PA 505 TL 326 DA 2285 SD 805 LP 500 KR 325 DS 2087 GI 804 VS 497 VF 324 WY 2079 NW 785 IR 493 MG 314 GD 2017 LS 760 YV 493 PN 313 GR 1985 LY 757 VY 478 RE 312 GL 1800 TY 749 IG 476 IV 311 DL 1777 PR 742 VT 475 KS 310 DF 1763 GE 737 TR 472 SC 310 GW 1725 SA 736 DN 471 FL 309 WS 1675 SF 728 SI 469 FF 306 AA 1671 PF 725 AD 462 CY 303 LD 1651 ND 693 LA 459 SH 302 EG 1610 ST 684 PP 451 LK 300 AG 1606 GH 683 RT 451 IT 298 RY 1558 YP 676 DT 448 LE 298 DP 1547 WL 675 RW 447 FS 296 GV 1500 SN 667 GQ 446 ED 294 RR 1498 TS 652 QG 446 RK 294 LG 1387 RD 648 TD 446 HF 292 GF 1386 YA 648 TA 437 VI 290 VG 1366 SL 644 TF 426 RH 287 GP 1339 RP 643 GK 422 MV 285 WF 1282 YL 638 YW 421 KY 284 FW 1277 IA 634 HD 420 Al 282 NY 1271 RL 627 IL 417 HS 281 PY 1209 EL 622 LV 406 YH 281 GT 1194 YN 607 IS 402 LN 278 WG 1177 AV 605 NG 398 PV 276 SR 1162 AP 600 RN 398 QY 276 TG 1142 AT 592 SV 397 WA 271 Dipeptides in HC CDR3s, part 2 QH 267 KD 176 II 102 NQ 53 FQ 264 SK 176 HI 101 CF 51 LI 257 YK 176 KP 101 MP 50 EV 255 EF 174 MY 100 CP 49 AM 253 FN 174 RM 99 RC 47 DQ 250 HN 171 AQ 98 HE 46 HR 250 FH 165 EQ 96 VC 46 PH 248 YQ 165 QT 96 QI 45 AN 242 KN 164 LM 95 MN 44 WR 242 MA 163 HV 94 MF 43 NF 240 NN 160 IK 93 HQ 41 PI 239 KA 159 PM 93 CD 38 TN 239 SQ 157 QN 93 CL 38 TI 238 PE 156 CG 91 NC 38 PW 229 WV 154 QF 91 HM 37 IP 228 EI 153 FI 90 FM 36 QR 227 TH 153 HW 90 ME 36 EW 225 FV 152 WH 90 MK 35 YI 221 AK 151 QV 89 QM 35 FE 220 TK 151 WI 89 NM 34 IY 220 WT 151 KH 88 KM 32 EP 219 PK 150 MI 88 TC 31 NR 217 KK 148 MS 87 CR 29 DM 214 IW 145 TQ 86 CV 25 FA 212 VH 145 NV 85 HC 25 AE 210 VE 141 EM 84 WM 25 IF 210 EE 138 HK 84 AC 24 QW 208 DE 136 IN 83 FC 24 YE 208 KL 136 NH 82 CA 23 FP 201 PQ 136 NI 82 CH 21 TM 201 QP 135 HT 81 CN 21 WE 201 SM 134 WK 79 MW 21 WP 201 QD 133 KF 77 PC 19 AH 199 QS 131 VM 73 LC 17 NP 198 VQ 130 MT 71 IC 16 VN 198 QQ 129 IH 69 MM 16 HA 196 WW 129 EH 68 MH 15 LH 196 NT 128 IE 67 WC 15 AW 193 DC 118 QK 65 EC 12 HP 192 KT 118 WQ 65 CK 10 HL 191 QA 118 GC 64 CW 10 RQ 191 NK 113 KE 61 MQ 10 TW 186 KW 112 KI 61 CI 9 EN 185 EK 109 CT 58 CC 8 LQ 182 FT 108 FK 58 CM 8 SE 180 KV 108 IM 57 CQ 6 VK 180 MR 105 KQ 57 QC 6 ET 178 TE 104 ML 55 CE 5 DK 177 HH 103 QE 55 KC 5 NL 177 IQ 103 NE 53 MC 3

TABLE 1060a Lengths of HC CDR3s vs which D segments occur (if any) for lengths 3-17 SEQ ID Length Name Sequence NO: 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1-1.1 GTTGT 156 0 0 0 0 0 0 2 2 3 6 2 3 3 4 0 1-1.2 VQLER 157 0 0 0 0 0 0 0 0 1 0 1 0 1 2 1 1-1.3 YNWND 158 0 0 0 0 0 2 6 14 16 19 16 14 17 16 9 1-7.1 GITGT 159 0 0 0 0 0 0 0 1 2 7 6 4 4 0 4 1-7.3 YNWNY 161 0 0 0 0 1 0 2 5 7 8 8 6 5 9 4 1-26.1 GIVGAT 164 0 0 0 1 0 0 2 4 10 4 6 9 3 2 2 1-26.2 V*WELL 165 0 0 0 0 0 0 0 0 1 0 1 0 0 0 1 1-26.3 YSGSYY 166 0 0 0 0 1 0 2 10 14 24 24 27 21 26 13 2-2.2 GYCSSTSCYT  70 0 0 0 0 0 0 0 0 0 2 2 9 15 15 11 2-2.3 DIVVVPAAI 168 0 0 0 0 0 0 0 0 0 0 1 3 2 5 5 2-8.2 GYCTNGVCYT 115 0 0 0 0 0 0 0 0 0 1 0 2 4 3 4 2-8.3 DIVLMVYAI 170 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 2-15.2 GYCSGGSCYS 136 0 0 0 0 0 0 0 0 1 3 5 12 10 25 22 2-15.3 DIVVVVAAT 172 0 0 0 0 0 0 0 0 0 0 1 1 2 3 1 2-21.2 AYCGGDCYS 174 0 0 0 0 0 0 0 0 0 1 1 3 5 2 5 2-21.3 HIVVVTAI 175 0 0 0 0 0 0 0 0 0 1 2 0 2 0 1 3-3.1 VLRFLEWLLY 176 0 0 0 0 0 0 0 0 0 0 1 2 2 2 3 3-3.2 YYDFWSGYYT 177 0 0 0 0 0 0 0 1 5 8 22 38 44 72 69 3-3.3 ITIFGVVII 178 0 0 0 0 0 0 0 0 0 0 0 1 0 2 5 3-9.1 VLRYFDWLL@ 179 0 0 0 0 0 0 0 0 1 0 0 4 5 5 5 3-9.2 YYDILTGYYN 180 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 3-10.1 VLLWFGELL@ 182 0 0 0 0 0 0 0 0 2 1 2 4 2 3 5 3-10.2 YYYGSGSYYN  81 0 0 0 0 0 0 0 2 4 7 10 13 15 18 14 3-10.2 ITMVRGVII 183 0 0 0 0 0 0 0 0 0 3 1 2 7 5 2 3-16.2 YYDYVWGSYRYT 104 0 0 0 0 0 0 0 0 0 0 0 1 7 7 7 3-16.2 IMITFGGVIVI 185 0 0 0 0 0 0 0 0 0 0 0 0 1 0 2 3-22.2 YYYDSSGYYY 187 0 0 0 0 0 0 0 0 6 30 45 56 59 108 101 3-22.3 ITMIVVVIT 188 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4-4.2 DYSNY 192 0 0 0 0 0 3 3 4 6 14 4 7 4 10 6 4-4.3 TTVT 190 0 0 0 0 2 4 11 19 23 19 25 19 10 11 9 4-17.2 DYGDY 195 0 0 2 6 12 8 38 40 48 47 50 40 29 21 10 4-23.2 DYGGNS 198 0 0 0 0 0 2 7 4 5 17 4 8 5 1 1 4-23.3 TTVVT 199 0 0 0 0 0 0 2 0 1 1 2 1 2 0 0 5-5.1 VDTAMV 200 0 0 0 0 0 0 0 1 4 8 1 3 2 0 1 5-5.2 WIQLWL 201 0 0 0 0 0 0 0 3 2 0 3 1 4 3 3 5-5.3 GYSYGY 202 0 0 0 0 1 6 9 20 43 29 27 22 32 26 27 5-12.1 VDIVATI 203 0 0 0 0 0 0 0 2 0 1 2 4 0 2 1 5-12.3 GYSGYDY 205 0 0 0 0 4 10 13 15 19 15 22 27 16 15 9 5-24.1 VEMATI 209 0 0 0 0 0 0 1 1 0 0 6 0 0 0 0 5-24.2 *RWLQL 210 0 0 0 0 1 0 3 1 3 2 2 1 2 2 2 5-24.3 RDGYNY 211 0 0 0 0 0 0 0 1 8 12 6 7 3 2 1 6-6.1 EYSSSS 212 0 0 0 0 0 0 0 9 7 16 19 13 2 4 2 6-6.2 SIAAR 213 0 0 0 1 1 0 17 8 7 13 17 6 16 16 7 6-6.3 V*QLV 214 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 6-13.1 GYSSSWY 215 0 0 0 0 0 1 6 11 35 40 56 44 50 42 35 6-13.2 GIAAAG 216 0 0 0 0 1 2 18 14 15 20 20 15 16 14 11 6-19.1 GYSSGWY 218 0 0 0 0 1 1 4 27 57 58 48 52 45 35 30 6-19.2 GIAVAG 219 0 0 0 0 1 1 0 7 8 20 8 13 16 8 10 6-19.3 V*QWLV 220 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 7-27.1 LTG 221 0 0 1 0 2 8 12 7 14 11 17 17 24 24 31 7-27.3 NWG 223 0 0 0 1 2 11 6 5 10 6 7 5 7 1 0 none 36 153 118 660 726 1007 2063 2463 1851 1596 1502 1075 874 681 609

TABLE 1060b Lengths of HC CDR3s vs which D segments occur (if any) for lengths 18-32 SEQ ID Length Name Sequence NO: 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 1-1.1 GTTGT 156 0 1 0 0 1 1 1 0 0 0 0 0 0 0 0 1-1.2 VQLER 157 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1-1.3 YNWND 158 6 5 3 4 1 1 1 0 0 1 0 0 0 0 0 1-7.1 GITGT 159 2 2 1 0 0 0 1 0 0 0 0 0 0 0 0 1-7.3 YNWNY 161 5 2 1 1 0 0 0 0 0 0 0 1 0 0 0 1-26.1 GIVGAT 164 0 0 3 0 1 0 0 1 0 0 0 0 0 0 0 1-26.2 V*WELL 165 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1-26.3 YSGSYY 166 14 11 9 8 6 3 3 0 3 1 0 0 0 0 0 2-2.2 GYCSSTSCYT  70 11 7 2 10 11 4 2 0 0 0 1 0 0 0 0 2-2.3 DIVVVPAAI 168 2 6 3 4 3 2 1 0 0 0 0 0 0 0 0 2-8.2 GYCTNGVCYT 115 3 0 1 1 3 1 0 0 0 0 0 0 0 0 0 2-8.3 DIVLMVYAI 170 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2-15.2 GYCSGGSCYS 136 20 10 7 4 8 9 3 0 0 0 0 0 0 0 0 2-15.3 DIVVVVAAT 172 0 1 1 1 0 0 0 0 1 0 0 0 0 0 0 2-21.2 AYCGGDCYS 174 1 3 1 1 1 0 0 0 0 0 0 0 0 0 0 2-21.3 HIVVVTAI 175 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3-3.1 VLRFLEWLLY 176 6 2 4 3 2 0 0 0 1 0 0 0 0 0 0 3-3.2 YYDFWSGYYT 177 82 97 104 67 61 32 23 7 3 4 0 0 2 1 0 3-3.3 ITIFGVVII 178 3 2 0 0 2 0 0 0 0 0 0 0 0 0 0 3-9.1 VLRYFDWLL@ 179 7 2 2 6 1 2 1 0 0 0 0 0 0 0 0 3-9.2 YYDILTGYYN 180 3 2 0 0 0 0 0 0 0 0 0 0 0 0 0 3-10.1 VLLWFGELL@ 182 3 4 0 0 0 0 0 0 0 0 0 0 0 0 0 3-10.2 YYYGSGSYYN  81 15 10 8 7 6 3 2 1 1 0 0 0 0 0 0 3-10.2 ITMVRGVII 183 3 3 6 0 0 0 0 0 0 0 0 0 0 0 0 3-16.2 YYDYVWGSYRYT 104 11 11 14 10 18 13 5 2 2 0 0 0 1 0 0 3-16.2 IMITFGGVIVI 185 1 3 1 0 0 0 0 0 0 0 0 0 0 0 0 3-22.2 YYYDSSGYYY 187 77 54 28 22 18 8 1 2 1 1 0 0 0 0 0 3-22.3 ITMIVVVIT 188 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 4-4.2 DYSNY 192 7 2 2 1 1 0 1 0 0 0 0 0 0 0 0 4-4.3 TTVT 190 4 2 2 1 3 0 0 0 0 0 1 0 0 0 0 4-17.2 DYGDY 195 17 7 8 3 2 1 1 0 1 0 1 0 0 0 0 4-23.2 DYGGNS 198 3 1 0 2 0 0 0 0 0 0 0 0 0 0 0 4-23.3 TTVVT 199 2 2 1 1 1 0 0 0 0 0 0 0 0 0 0 5-5.1 VDTAMV 200 0 1 1 2 1 0 0 0 0 0 0 0 0 0 0 5-5.2 WIQLWL 201 3 3 1 0 1 0 0 0 0 1 0 0 0 1 0 5-5.3 GYSYGY 202 13 18 7 2 2 6 0 0 2 0 0 0 0 0 0 5-12.1 VDIVATI 203 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 5-12.3 GYSGYDY 205 11 10 6 6 0 2 0 0 0 0 0 0 0 0 0 5-24.1 VEMATI 209 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 5-24.2 *RWLQL 210 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5-24.3 RDGYNY 211 2 2 0 0 0 0 0 0 0 0 0 0 0 0 0 6-6.1 EYSSSS 212 9 3 1 1 0 0 0 1 0 0 0 0 0 0 0 6-6.2 SIAAR 213 2 3 2 6 0 0 0 0 0 0 0 0 0 0 0 6-6.3 V*QLV 214 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6-13.1 GYSSSWY 215 24 22 17 4 5 1 3 2 0 0 1 0 0 0 0 6-13.2 GIAAAG 216 10 4 2 5 2 1 0 0 0 0 0 0 0 0 0 6-19.1 GYSSGWY 218 23 25 13 7 7 3 0 2 1 0 1 0 0 0 0 6-19.2 GIAVAG 219 6 2 2 0 0 0 2 0 0 0 0 0 0 0 0 6-19.3 V*QWLV 220 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 7-27.1 LTG 221 29 23 12 9 7 4 3 1 0 1 0 0 0 0 0 7-27.3 NWG 223 1 0 0 0 2 0 0 0 0 0 0 0 0 0 0 none 339 217 198 85 60 36 27 13 9 2 1 1 0 0 1

TABLE 1082 Tally of J stumps ---AEYFQHWGQGTLVTSS 6.15% (SEQ ID NO: 66) JH1 A C D E F G H I K L M N P Q R S T V W Y — 4   41  0    0  0    0    0   0    0  0    0    0   0   0   0  0   0  0    0   0    0  605 5    1  0    1 64    0    6   0    1  0    0    0   0   0   1  2   1  1    0   0    0  568 6    0  0     0  0    0    1   1    0  0    3    0   2   1   0  1   1  0    0   0  211  425 7    1  0    1  0  363    3   1    0  0    8    0   0   1   0  1   6  0    3   0    6  252 8    8  0   59 23    4   17  11    3  5   19    0   8   3 221 11   6  8    5   0    4  231 9    2  1   13  2   13    3 447   19  2    6    3  20   1   2  0  20  3    5   4    9   71 ---YWYFDLWGRGTLVTVSS 7.80% (SEQ ID NO: 67) JH2 A C D E F G H I K L M N P Q R S T V W Y — 4    0  0    0  0    0    0   0    0  0    0    0   0   0   0  0   0  0    0   0  159 1519 5    0  0    2  0    2   11   0    0  0    1    0   1   0   0  7   1  1    1 929   21  701 6    3  2    9  0   40    5   7    4  1    9    0   7   1   0  1  11  2    0   1 1083  492 7    1  6    1  0 1209    2   6   23  0   89   30   1   2   1  1  12  0     11   1   42  240 8   31  2 1241 90    4   38  30    3  1    0    1  29   3   4  2   2  3   19   0   15  160 9    3  1    9  3   34    2  26   17  5 1064   36  17  30  38  33  20  8   83   1  177   71 -----AFDIWGQGTMVTVSS 25.4% (SEQ ID NO: 2) JH3 A C D E F G H I K L M N P Q R S T V W Y — 6 4374  0    0  0    0    0  0    0  0    0    0   0   0   0   0   0  0    0   0    0 1074 7    1  4    0  1 4612    2  0    8  0   56    3   2   1   1   0  29  2   15  12   74  625 8   23  0 4765 51    0   28 14    0  3    0    0  15   2   1   1   2  2    6   0    4  531 9    7  5    5  0   73 2  1 4439  4   64   64  43   2   1  11  54 49  113   2   18  491 -----YFDYWGQGTLVTVSS 34.7% (SEQ ID NO: 1) JH4 A C D E F G H I K L M N P Q R S T V W Y — 6    0  0    0  0    0    0   0    0  0    0    0   0   0   0   0   0  0    0   0 1975 5683 7    9  1   10  7 3950   31   6   26  0  109    4   5  24   4   5  35  7   28  16   59 3322 8   26  0 5991 32    5   91  19    2  1    6    1  21   7   0   8  13  3    6   2   14 1410 9    5 18   17  0  119    2   0    0 14    0    2  64  15  16  10 216 11    3   6 6317  823 ----NWFDPWGQGTLVTVSS 4.8% (SEQ ID NO: 68) JH5 A C D E F G H I K L M N P Q R S T V W Y — 5    0  0    0  0    0    0   0    0  0    0    0 274   0   0   0   0  0    0   0    0  764 6    2  1    0  0    0    1   0    0  0    0    0   0   0   0   0   0  0    0 644      0  390 7    1  1    0  0  768    2   1    1  0   13    0   0   0   0   0   1  0    2   0    4  244 8    2  0  810  3    0    1   0    0  0    0    0   1   0   0   0   0  1    1   0    1  218 9    3  0    3  0    3    0   3    0  0    4    0   4 814   1   0  14  2    0   0    0  187 YYYYYGMDVWGQGTTVTVSS 21.1% (SEQ ID NO: 3) JH6 A C D E F G H I K L M N P Q R S T V W Y — 1    0  0    0  0    0    0  0    0  0    0    0   0   0   0   0   0  0    0   0  690 3967 2    4  0   15  0   16   31 12    2  1    4    0   8   7   4  14  18  7    4   1 1694 2815 3    3  0   19  3   14   23 16    1  5    9    0  16   9   5  12  20  4    1   4 2440 2053 4    4  0   14  2   31   16 25    3  2   10    0  13   6   5  15  35  5    5   4 2815 1647 5    2  1    9  1   23   19 21    1  1    3    1  15   3   1   7  26  3    0   1 3350 1169 6   69  5   14  1    4 3057  8    1  0    0    0   8   7   5   4  15  3    7   0  657  792 7    3  0    1  0    4    5  0   18  2  108 3866   0   2   2   3   1  7   18   3    1  613 8    7  0 4064  5    1   17  4    1  0    2    0  11   2   0   0   3  2    3   2    6  527 9    9  0    1  0    6    1  1   19  0    7    2   1   1   0   1   4  0 4092   0    1  511

TABLE 1097 HC CDR3 libraries CDR3 D region DJ fill J stump Library Length Leadin sequence sequence sequence FR4 sequence 5.001 10, 11, 12 6, 7, or 8 none none AFDI WGQGTMVTVSS X (diversity (JH4) (SEQ X = (1.0G, in text) ID NO: 987) .57S, (SEQ ID .46R, NO: 986) .42D, .36L, .35Y) 5.002 13, 14, 15 6, 7, 8 X none none YYYGMDV WGQGTTVTVSS X = (1.0G, (diversity (jh6) (SEQ .57S, in text) ID NO: 988) .46R, (SEQ ID .42D, NO: 984) .36L, .35Y) 5.003 18, 19, 20 4, 5, 6 X = YYDFWSGYYT none YFDY WGQGTLVTVSS (1.0G, (3-3.2) (SEQ ID (JH3) (SEQ .57S, (SEQ ID NO: 985) ID NO: 989) .46R, NO: 177) .42D, .36L, .35Y) 5.004 15, 16, 17 2, 3, 4 X = GYSSGWY 2X, X = AFDI WGQGTMVTVSS (1.0G, (6-19.1) (1.0G, (SEQ ID (JH4) (SEQ .57S, (SEQ ID .57S, NO: 986) ID NO: 987) .46R, NO: 218) .46R, .42D, .42D, .36L, .36L, .35Y) .35Y) 5.005 11-14 0, 1, 2 X = GYSSGWY 0, 1 X = YFDY WGQGTLVTVSS (1.0G, (6-19.1) (1.0G, (SEQ ID (JH3) (SEQ .57S, (SEQ ID .57S, NO: 985) ID NO: 989) .46R, NO: 218) .46R, .42D, .42D, .36L, .36L, .35Y) .35Y) 5.006 19-25 0, 1, 2, 3 X GYCSGGSCYS 0, 1, 2, 3 YYYYYGMDV WGQGTTVTVSS X = (1.0G, (2-2.2) X = (1.0G, (parent AA (jh6) (SEQ .57S, (Cys .57S, 8X others) ID NO: 988) .46R, residues .46R, (SEQ ID .42D, constant) .42D, NO: 990) .36L, (SEQ ID .36L, .35Y) NO: 136) .35Y)

REFERENCES

The contents of all cited references including literature references, issued patents, published or non-published patent applications cited throughout this application as well as those listed below are hereby expressly incorporated by reference in their entireties. In case of conflict, the present application, including any definitions herein, will control.

-   U.S. Published Application 2005-0119455A1 -   Sidhu et al., J Mol Biol. 2004 338:299-310.

EQUIVALENTS

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. 

The invention claimed is:
 1. A library of vectors or genetic packages, wherein the vectors or genetic packages comprise variegated DNA sequences, each encoding a heavy chain (HC) variable region, which comprises three complementarity determining regions (CDRs) 1-3 and framework regions (FRs) 1-4, arranged from the amino-terminus to the carboxy-terminus in an order of FR1-CDR2-FR2-CDR2-FR3-CDR3-FR4, wherein the HC CDR3 region contains 3-35 amino acid residues, and wherein more than 20% of the HC CDR3 sequence consists of Tyr residues; wherein D regions and J stumps of the HC CDR3 consist of more than 20% of Tyr residues; wherein the D regions are selected from the group consisting of 3-3.2 (SEQ ID NO: 177), 3-22.2 (SEQ ID NO: 88), 6-19.1 (SEQ ID NO: 218), 6-13.1 (SEQ ID NO: 215) and 4-17.2 (SEQ ID NO: 760); and wherein the J stumps are selected from the libraries consisting of 5.002 (SEQ ID NO: 984), 5.003 (SEQ ID NO: 985), 5.005 (SEQ ID NO: 985) and 5.006 (SEQ ID NO: 990).
 2. The library of claim 1, wherein the library is a library of genetic packages.
 3. The library of claim 2, wherein the genetic packages are bacteriophages.
 4. The library of claim 3, wherein the library is a phage display library.
 5. The library of claim 1, wherein the library is a library of vectors.
 6. The library of claim 5, wherein the vectors are phage vectors or phagemid vectors.
 7. The library of claim 1, wherein the library comprises at least 1×10⁹ members. 